TNF-alpha converting enzyme

ABSTRACT

A metalloprotease that converts TNF-α from the 26 kD cell form to the 17 kD form has been isolated and purified and the cDNA sequence known. In particular, the protease has a molecular weight of approximately 80 kD. The isolated and purified protease is useful for designing an inhibitor thereof, and may find use as a therapeutic agent Assays for detecting the protease-inhibiting activity of a molecule are also an aspect of the invention.

[0001] This application claims priority under 35 U.S.C. §119(e)(1) ofSer. No. 08/504,614, filed Jul. 20, 1995 and of Ser. No. 08/428,458,filed Jun. 8, 1995.

FIELD OF THE INVENTION

[0002] The invention is directed to purified and isolated TNF-αconverting enzyme, the nucleic acids encoding such enzyme, processes forproduction of recombinant TNF-α convertases, pharmaceutical compositionscontaining such enzymes, and their use in various assays and therapies.

BACKGROUJND OF THE INVENTION

[0003] Tumor necrosis factor-α (TNF-α, also known as cachectin) is amammalian protein capable of inducing a variety of effects on numerouscell types. TNF-α was initially characterized by its ability to causelysis of tumor cells and is produced by activated cells such asmononuclear phagocytes;T-cells, B-cells, mast cells and NK cells. Thereare two forms of TNF-α, a type II membrane protein of relative molecularmass 26,000 (26 kD) and a soluble 17 kD form generated from thecell-bound protein by proteolytic cleavage. TNF-α is a principalmediator of the host response to gram-negative bacteria.Lipopolysaccharide (LPS, also called endotoxin), derived from the cellwall of gram-negative bacteria, is a potent stimulator of TNF-αsynthesis. Because the deleterious effects which can result from anover-production or an unregulated-production of TNF-α are extremelyserious, considerable efforts have been made to control or regulate theserum level of TNF-α. An important part in the effort to effectivelycontrol serum TNF-α levels is the understanding of the mechanism ofTNF-α biosynthesis.

[0004] The mechanism by which TNF-α is secreted has not previously beenelucidated. Kriegler et al. Cell, 53:45 (1988) conjectured that TNF-α“secretion” is due to the converting of the 26 k membrane-bound moleculeby a then unknown proteolytic enzyme or protease. Scuderi et. al., J.Immunology, 143:168 (1989), suggested that the release of TNF-α fromhuman leukocyte cells is dependent on one or more serine proteases,e.g., a leukocyte elastase or trypsin. A serine protease inhibitor,p-toluenesulfonyl-L-arginine methyl ester, was found to suppress humanleukocyte TNF-α release in a concentration-dependent manner. Scuderi et.al. suggested that an arginine methyl ester competes for thearginine-binding site in the enzyme's reactive center and thereby blockshydrolysis. The lysine and phenylalanine analogs of the inhibitorreportedly failed to mimic the arginine methyl ester. However, it wasnever shown that this compound acted by inhibiting a protease thatcleaves the 26 kD TNF. More recently, it has been reported thatmetalloprotease inhibitors block the release of TNF from THP-1 cells.See Mohler et al., Nature 370:218 (1994); Gearing et al., Nature,370:555 (1994); and McGeehan et al., Nature, 370:568 (1994).

[0005] Most, but not all, proteases recognize a specific amino acidsequence. Some proteases primarily recognize residues located N-terminalof the cleaved bond, some recognize residues located C-terminal of thecleaved bond, and some proteases recognize residues on both sides of thecleaved bond. Metalloprotease enzymes utilize a bound metal ion,generally Zn²⁺, to catalyze the hydrolysis of the peptide bond.Metalloproteases are implicated in joint destruction (the matrxmetalloproteases), blood pressure regulation (angiotensin convertingenzyme), and regulation of peptide-hormone levels (neutralendopeptidase-24.11).

SUMMARY OF THE INVENTION

[0006] The invention pertains to biologically active TNF-α convertingenzyme (“TACE”) as an isolated and purified polypeptide. In addition,the invention is directed to isolated nucleic acids encoding TACE and toexpression vectors comprising a cDNA encoding TACE. Within the scope ofthis invention are host cells that have been transfected or transformedwith expression vectors that comprise a cDNA encoding TACE, andprocesses for producing TACE by culturing such host cells underconditions conducive to expression of TACE. By virtue of thepurification of TACE, antibodies, and in particular, monoclonalantibodies against TACE are an aspect of the invention. In addition,assays utilizing TACE to screen for potential inhibitors thereof, andmethods of using TACE as a therapeutic agent for the treatment ofdiseases mediated by cell-bound TNF-α or other molecules are encompassedby the invention. Further, methods of using TACE in the design ofinhibitors thereof are also an aspect of the invention.

[0007] The isolated and purified metalloprotease of the invention iscapable of converting TNF-α from the 26 kD membrane-bound form to the 17kD form, and which has a molecular weight of between approximately 66 kDand approximately 97 kD. The cDNA sequence of TACE is shown in SEQ IDNO:1. The isolated and purified TNF-α converting enzyme (“TACE”)comprises amino acids 18-824 of SEQ ID NO:2.

[0008] Inhibition of the TACE inhibits release of TNf-α into the serumand other extracellular spaces. TACE inhibitors would therefore haveclinical utility in treating conditions characterized by over-productionor upregulated production of TNF-α. A particularly useful TACE inhibitorfor certain pathological conditions would selectively inhibit TACE whilenot affecting TNF-β (also known as lymphotoxin) serum levels. Theover-production or unregulated production of TNF-α has been implicatedin certain conditions and diseases, for example, Systemic InflammatoryResponse Syndrome, reperfusion injury, cardiovascular disease,infectious disease such as HIV infection and HIV neuropathy, obstetricalor gynecological disorders, inflammatory disease/auto-immunity,allergic/atopic diseases, malignancy, transplants including organtransplant rejection or graft-versus-host disease, cachexia, congenital,dermatologic, neurologic, renal, toxicity, and metabolic/idiopathicdiseases.

[0009] Inhibitors of TACE would prevent the cleavage of cell-bound TNF-αthereby reducing the level of TNF-α in serum and tissues. The presentinvention encompasses such an embodiment and comprises a method ofinhibiting the cleavage of TNF-α from cell membranes in a mammalcomprising administering to such mamma an effective amount of a compoundthat inhibits the TNF-α proteolytic activity of an enzyme comprising thesequence of amino acids from 18 to 671 through 824 of SEQ ID NO:2. Inaddition, the invention comprises a method for treating a mammal havinga disease characterized by an overproduction or an upregulatedproduction of TNF-α, comprising administering to the mammal acomposition comprising an effective amount of a compound that inhibitsthe TNF-α proteolytic activity of an enzyme comprising the sequence ofamino acids 18-824 of SEQ ID NO:2. Such inhibitors would be ofsignificant clinical utility and could be potential therapeutics fortreating the above-listed TNF-α-related disorders. Isolation andpurification of TACE would provide a significant advancement in theeffort to develop inhibitors of such enzyme, and the treatment ofTNF-associated diseases, and indeed, could lead to use of TACE itself asa therapeutic agent for certain physiological disorders. For example, inaddition to TNF-α, other cytokines as well as cytokine receptors andseveral adhesion proteins may be released from the cell surface by TACEor related proteases. TACE may be administered to modulate or removecell surface cytokines, cytokine receptors and adhesion proteinsinvolved in tumor cell growth, inflammation, or fertilization.

DETAILED DESCRIPTION OF THE INVENTION

[0010] A cDNA encoding human TNF-α converting enzyme (“TACE”) has beenisolated and is disclosed in SEQ ID NO:1. This discovery of the cDNAencoding human TACE enables construction of expression vectorscomprising nucleic acid sequences encoding TACE; host cells transfectedor transformed with the expression vectors; biologically active humanTACE as isolated and purified proteins; and antibodies imnmunoreactivewith TACE.

[0011] Isolated and purified TACE polypeptides according to theinvention are useful for detecting the TACE-inhibiting activity of amolecule. In such a method involving routine and conventionaltechniques, a molecule of unknown TACE-inhibiting activity is mixed witha substrate and incubated with a TACE polypeptide. The extent ofsubstrate cleavage then can be determined chromatographically.

[0012] In addition, TACE polypeptides according to the invention areuseful for the structure-based design of a TACE inhibitor. Such a designwould comprise the steps of determining the three-dimensional structureof such TACE polypeptide, analyzing the three-dimensional dimensionalstructure for the likely binding sites of substrates, synthesizing amolecule that incorporates a predictive reactive site, and determiningthe TACE-inhibiting activity of the molecule.

[0013] Antibodies immunoreactive with TACE, and in particular,monoclonal antibodies against TACE, are now made available through theinvention. Such antibodies may be useful for inhibiting TACE activity invivo and for detecting the presence of TACE in a sample.

[0014] As used herein, the term “TACE” refers to a genus of polypeptidesthat are capable of converting the 26 kD cell membrane-bound form ofTNF-α (that includes an intracellular region, a membrane region, and anextracellular region), into the soluble 17 kD form that comprises theC-terminal 156 residues of the TNF-α protein. TACE encompasses proteinshaving the amino acid sequence 18 to 824 of SEQ ID NO:2, as well asthose proteins having a high degree of similarity (at least 80%, andmore preferably 90% homology) with the amino acid sequence 18 to 824 ofSEQ ID NO:2 and which proteins are biologically active. In addition,TACE refers to the biologically active gene products of the nucleotides52-2472 of SEQ ID NO:1. Further encompassed by the term “TACE” are themembrane-bound proteins (which include an intracellular region, amembrane region, and an extracellular region), and soluble or truncatedproteins which comprise primarily the extracellular portion of theprotein, retain biological activity and are capable of being secreted.Specific examples of such soluble proteins are those comprising thesequence of amino acids 18-671 of SEQ ID NO:2. Truncated versions arethose having less than the extracellular portion of the protein andcomprise, for example, amino acids 18-477 of SEQ ID NO:2, or thatcomprise substantially all of the catalytic domain, i.e., amino acids215 to 477 of SEQ ID NO:2.

[0015] The isolated and purified TACE according to the invention has amolecular weight between about 66 kD and about 97 kD as determined bySDS-polyacrylamide gel electrophoresis (SDS-PAGE). More specifically,TACE was found to have a molecular weight of approximately 80 kD asdetermined by SDS-PAGE.

[0016] The term “isolated and purified” as used herein, means that TACEis essentially free of association with other proteins or polypeptides,for example, as a purification product of recombinant host cell cultureor as a purified product from a non-recombinant source. The term“substantially purified” as used herein, refers to a mixture thatcontains TACE and is essentially free of association with other proteinsor polypeptides, but for the presence of known proteins that can beremoved using a specific antibody, and which substantially purified TACEretains biological activity. The term “purified TACE” refers to eitherthe “isolated and purified” form of TACE or the “substantially purified”form of TACE, as both are described herein.

[0017] The term “biologically active” as it refers to TACE, means thatthe TACE is capable of converting the 26 kD cell form of TNF-α into the17 cD form.

[0018] A “nucleotide sequence” refers to a polynucleotide molecule inthe form of a separate fragment or as a component of a larger nucleicacid construct, that has been derived from DNA or RNA isolated at leastonce in substantially pure form (i.e., free of contaminating endogenousmaterials) and in a quantity or concentration enabling identification,manipulation, and recovery of its component nucleotide sequences bystandard biochemical methods (such as those outlined in Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring HarborLaboratiry, Cold Spring Harbor, N.Y. (1989)). Such sequences arepreferably provided in the form of an open reading frame uninterruptedby internal non-translated sequences, or introns, that are typicallypresent in eukaryotic genes. Sequences of non-translated DNA may bepresent 5′ or 3′ from an open reading frame, where the same do notinterfere with manipulation or expression of the coding region.

[0019] A “TACE variant” as referred to herein, means a polypeptidesubstantially homologous to native TACE, but which has an amino acidsequence different from that of native TACE (human, murine or othermammalian species) because of one or more deletions, insertions orsubstitutions. The variant amino acid sequence preferably is at least80% identical to a native TACE amino acid sequence, most preferably atleast 90% identical. The percent identity may be determined, forexample, by comparing sequence information using the GAP computerprogram, version 6.0 described by Devereux et al. (Nucl. Acids Res.12:387, 1984) and available from the University of Wisconsin GeneticsComputer Group (UWGCG). The GAP program utilizes the alignment method ofNeedleman and Wunsch (J. Mol. Biol. 48:443, 1970), as revised by Smithand Waterman (Adv. Appl. Math 2:482, 1981). The preferred defaultparameters for the GAP program include: (1) a unary comparison matrix(containing a value of 1 for identities and 0 for non-identities) fornucleotides, and the weighted comparison matrix of Gribskov and Burgess,Nucl. Acids Res. 14:6745, 1986, as described by Schwartz and Dayhoff,eds., Atlas of Protein Sequence and Structure, National BiomedicalResearch Foundation, pp. 353-358, 1979; (2) a penalty of 3.0 for eachgap and an additional 0.10 penalty for each symbol in each gap; and (3)no penalty for end gaps.

[0020] Variants may comprise conservatively substituted sequences,meaning that a given amino acid residue is replaced by a residue havingsimilar physiochemical characteristics. Conservative substitutions arewell known in the art and include substitution of one aliphatic residuefor another, such as Ile, Val, Leu, or Ala for one another, orsubstitutions of one polar residue for another, such as between Lys andArg, Glu and Asp; or Gln and Asn. Conventional procedures and methodscan be used for making and using such variants. Other such conservativesubstitutions, for example, substitutions of entire regions havingsimilar hydrophobicity characteristics, are well known and routinelyperformed. Naturally occurring TACE variants are also encompassed by theinvention. Examples of such variants are proteins that result fromalternate mRNA splicing events or from proteolytic cleavage of the TACEprotein, wherein the TACE proteolytic property is retained. Alternatesplicing of mRNA may yield a truncated but biologically active TACEprotein, such as a naturally occurring soluble form of the protein, forexample, as shown in SEQ ID NO:4. Variations attributable to proteolysisinclude, for example, differences in the N- or C-termini upon expressionin different types of host cells, due to proteolytic removal of one ormore terminal amino acids from the TACE protein (generally from 1-5terminal amino acids).

[0021] As stated above, the invention provides isolated and purified, orhomogeneous, TACE polypeptides, both recombinant and non-recombinant.Variants and derivatives of native TACE proteins that retain the desiredbiological activity may be obtained by mutations of nucleotide sequencescoding for native TACE polypeptides. Alterations of the native aminoacid sequence may be accomplished by any of a number of conventionalmethods. Mutations can be introduced at particular loci by synthesizingoligonucleotides containing a mutant sequence, flanked by restrictionsites enabling ligation to fragments of the native sequence. Followingligation, the resulting reconstructed sequence encodes an analog havingthe desired amino acid insertion, substitution, or deletion.

[0022] Alternatively, oligonucleotide-directed site-specific mutagenesisprocedures can be employed to provide an altered gene whereinpredetermined codons can be altered by substitution, deletion orinsertion. Exemplary methods of making the alterations set forth aboveare disclosed by Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene37:73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al.(Genetic Engineering: Principles and Methods, Plenum Press, 1981);Kunkel (Proc. Natl. Acad. Sci. USA 82:488, 1985); Kunkel et al. (Methodsin Enzymol. 154:367, 1987); and U.S. Pat. Nos. 4,518,584 and 4,737,462all of which are incorporated by reference.

[0023] TACE may be modified to ceate TACE derivatives by formingcovalent or aggregative conjugates with other chemical moieties, such asglycosyl groups, polyethylene glycol (PEG) groups, lipids, phosphate,acetyl groups and the like. Covalent derivatives of TACE may be preparedby linking the chemical moieties to functional groups on TACE amino acidside chains or at the N-terminus or C-terminus of a TACE polypeptde orthe extracellular domain thereof. Other derivatives of TACE within thescope of this invention include covalent or aggregative conjugates ofTACE or its fragments with other proteins or polypeptides, such as bysynthesis in recombinant culture as N-termninal or C-terminal fusions.For example, the conjugate may comprise a signal or leader polypeptidesequence (e.g. the α-factor leader of Saccharomyces) at the N-terminusof a TACE polypeptide. The signal or leader peptide co-translationallyor post-translationally directs transfer of the conjugate from its siteof synthesis to a site inside or outside of the cell membrane or cellwall.

[0024] TACE polypeptide conjugates can comprise peptides added tofacilitate purification and identification of TACE. Such peptidesinclude, for example, poly-His or the antigenic identification peptidesdescribed in U.S. Pat. No. 5,011,912 and in Hopp et al., Bio/Technology6:1204, 1988.

[0025] The invention further includes TACE polypeptides with or withoutassociated native-pattern glycosylation. TACE expressed in yeast ormammalian expression systems (e.g., COS-7 cells) may be similar to orsignificantly different from a native TACE polypeptide in molecularweight and glycosylation pattern, depending upon the choice ofexpression system. Expression of TACE polypeptides in bacterialexpression systems, such as E. coli, provides non-glycosylatedmolecules. Glycosyl groups may be removed through conventional methods,in particular those utilizing glycopeptidase. In general, glycosylatedTACE may be incubated with a molar excess of glycopeptidase (BoehringerMannheim).

[0026] Equivalent DNA constructs that encode various additions orsubstitutions of amino acid residues or sequences, or deletions ofterminal or internal residues or sequences not needed for biologicalactivity are encompassed by the invention. For example, N-glycosylationsites in the TACE extracellular domain can be modified to precludeglycosylation, allowing expression of a reduced carbohydrate analog inmammalian and yeast expression systems. N-glycosylation sites ineukaryotic polypeptides are characterized by an amino acid tripletAsn-X-Y, wherein X is any amino acid except Pro and Y is Ser or Thr.Appropriate substitutions, additions or deletions to the nucleotidesequence encoding these triplets will result in prevention of attachmentof carbohydrate residues at the Asn side chain. Alteration of a singlenucleotide, chosen so that Asn is replaced by a different amino acid,for example, is sufficient to inactivate an N-glycosylation site. Knownprocedures for inactivating N-glycosylation sites in proteins includethose described in U.S. Pat. No. 5,071,972 and EP 276,846, herebyincorporated by reference.

[0027] In another example, sequences encoding Cys residues that are notessential for biological activity can be altered to cause the Cysresidues to be deleted or replaced with other amino acids, preventingformation of incorrect intramolecular disulfide bridges uponrenaturation. Other equivalents are prepared by modification of adjacentdibasic amino acid residues to enhance expression in yeast systems inwhich KEX2 protease activity is present. EP 212,914 discloses the use ofsite-specific mutagenesis to inactivate KEX2 protease processing sitesin a protein. KEX2 protease processing sites are inactivated bydeleting, adding or substituting residues to alter Arg-Arg, Arg-Lys, andLys-Arg pairs to eliminate the occurrence of these adjacent basicresidues. Lys-Lys pairings are considerably less susceptible to KEX2cleavage, and conversion of Arg-Lys or Lys-Arg to Lys-Lys represents aconservative and preferred approach to inactivating KEX2 sites.

[0028] Nucleic acid sequences within the scope of the invention includeisolated DNA and RNA sequences that hybridize to the native TACEnucleotide sequences disclosed herein under conditions of moderate orhigh stringency, and which encode biologically active TACE. Conditionsof moderate stringency, as known to those having ordinary skill in theart, and as defined by Sambrook et al. Molecular Cloning: A LaboratoryManual, 2 ed. Vol. 1, pp. 1.101-104, Cold Spring Harbor LaboratoryPress, (1989), include use of a prewashing solution of 5 X SSC, 0.5%SDS, 1.0 mM EDTA (pH 8.0) and hybridization conditions of about 50°C.-60° C., 5 X SSC, overnight, preferably 55° C. Conditions of highstringency include higher temperatures of hybridization and washing. Theskilled artisan will recognize that the temperature and wash solutionsalt concentration may be adjusted as necessary according to factorssuch as the length of the probe.

[0029] Due to the known degeneracy of the genetic code wherein more thanone codon can encode the same amino acid, a DNA sequence may vary fromthat shown in SEQ ID NO: I and still encode a TACE protein having theamino acid sequence of SEQ ID NO:2. Such variant DNA sequences mayresult from silent mutations (e.g., occurring during PCR amplification),or may be the product of deliberate mutagenesis of a native sequence.

[0030] The invention thus provides equivalent isolated DNA sequencesencoding biologically active TACE, selected from: (a) the coding regionof a native mammalian TACE gene; (b) cDNA comprising the nucleotidesequence presented in SEQ ID NO:1; (c) DNA capable of hybridization to aDNA of (a) under moderately stringent conditions and which encodesbiologically active TACE; and (d) DNA which is degenerate as a result ofthe genetic code to a DNA defined in (a), (b) or (c) and which encodesbiologically active TACE. TACE proteins encoded by such DNA equivalentsequences are encompassed by the invention.

[0031] DNA that are equivalents to the DNA sequence of SEQ ID NO:1 willhybridize under moderately stringent or highly stringent conditions tothe double-stranded native DNA sequence that encode polypeptidescomprising amino acid sequences of 18-Xaa of SEQ ID NO:2, wherein Xaa isan amino acid from 671 to 824. Examples of TACE proteins encoded by suchDNA, include, but are not limited to, TACE fragments (soluble ormembrane-bound) and TACE proteins comprising inactivated N-glycosylationsite(s), inactivated KEX-2 protease processing site(s), or conservativeamino acid substitution(s), as described above. TACE proteins encoded byDNA derived from other mammalian species, wherein the DNA will hybridizeunder conditions of moderate or high stringency to the complement of thecDNA of SEQ ID NO:1 are also encompassed.

[0032] Alternatively, TACE-binding proteins, such as the anti-TACEantibodies of the invention, can be bound to a solid phase such as acolumn chromatography matrix or a similar substrate suitable foridentifying, separating or purifying cells that express the TACE ontheir surface. Adherence of TACE-binding proteins to a solid phasecontacting surface can be accomplished by any means, for example,magnetic microspheres can be coated with TACE-binding proteins and heldin the incubation vessel through a magnetic field. Suspensions of cellmixtures are contacted with the solid phase that has TACE-bindingproteins thereon. Cells having TACE on their surface bind to the fixedTACE-binding protein and unbound cells then are washed away. Thisaffinity-binding method is useful for purifying, screening or separatingsuch TACE-expressing cells from solution. Methods of releasingpositively selected cells from the solid phase are known in the art andencompass, for example, the use of enzymes. Such enzymes are preferablynon-toxic and non-injurious to the cells and are preferably directed tocleaving the cell-surface binding partner.

[0033] Alternatively, mixtures of cells suspected of containingTACE-expressing cells first can be incubated with a biotinylatedTACE-binding protein. Incubation periods are typically at least one hourin duration to ensure sufficient binding to TACE. The resulting mixturethen is passed through a column packed with avidin-coated beads, wherebythe high affinity of biotin for avidin provides the binding of theTACE-binding cells to the beads. Use of avidin-coated beads is known inthe art. See Berenson, et al. J. Cell. Biochem., 10D:239 (1986). Wash ofunbound material and the release of the bound cells is performed usingconventional methods.

[0034] In the methods described above, suitable TACE-binding proteinsare anti-TACE antibodies, and other proteins that are capable ofhigh-affinity binding of TACE. A preferred TACE-binding protein is ananti-TACE monoclonal antibody obtained, for example, as described inExample 4.

[0035] TACE polypeptides may exist as oligomers, such ascovalently-linked or non-covalently-linked dimers or trimers. Oligomersmay be linked by disulfide bonds formed between cysteine residues ondifferent TACE polypeptides. In one embodiment of the invention, a TACEdimer is created by fusing TACE to the Fc region of an antibody (e.g.,IgG1) in a manner that does not interfere with biological activity ofTACE. The Fc polypeptide preferably is fused to the C-terminus of asoluble TACE (comprising only the extracellular domain). Generalpreparation of fusion proteins comprising heterologous polypeptidesfused to various portions of antibody-derived polypeptides (includingthe Fe domain) has been described, e.g., by Ashkenazi et al. (PNAS USA88:10535, 1991) and Byrn et al. (Nature 344:677, 1990), herebyincorporated by reference. A gene fusion encoding the TACE:Fc fusionprotein is inserted into an appropriate expression vector. TACE:Fcfusion proteins are allowed to assemble much like antibody molecules,whereupon interchain disulfide bonds form between Fc polypeptides,yielding divalent TACE. If fusion proteins are made with both heavy andlight chains of an antibody, it is possible to form a TACE oligomer withas many as four TACE extracellular regions. Alternatively, one can linktwo soluble TACE domains with a peptide linker.

[0036] Recombinant expression vectors containing a nucleic acid sequenceencoding TACE can be prepared using well known methods. The expressionvectors include a TACE DNA sequence operably linked to suitabletranscriptional or translational regulatory nucleotide sequences, suchas those derived from a mammalian, microbial, viral, or insect gene.Examples of regulatory sequences include transcriptional promoters,operators, or enhancers, an mRNA ribosomal binding site, and appropriatesequences which control transcription and translation initiation andtermination. Nucleotide sequences are “operably linked” when theregulatory sequence functionally relates to the TACE DNA sequence. Thus,a promoter nucleotide sequence is operably linked to a TACE DNA sequenceif the promoter nucleotide sequence controls the transcription of theTACE DNA sequence. The ability to replicate in the desired host cells,usually conferred by an origin of replication, and a selection gene bywhich transformants are identified, may additionally be incorporatedinto the expression vector.

[0037] In addition, sequences encoding appropriate signal peptides thatare not naturally associated with TACE can be incorporated intoexpression vectors. For example, a DNA sequence for a signal peptide(secretory leader) may be fused in-frame to the TACE sequence so thatTACE is initially translated as a fusion protein comprising the signalpeptide. A signal peptide that is functional in the intended host cellsenhances extracellular secretion of the TACE polypeptide. The signalpeptide may be cleaved from the TACE polypeptide upon secretion of TACEfrom the cell.

[0038] Suitable host cells for expression of TACE polypeptides includeprokaryotes, yeast or higher eukaryotic cells. Appropriate cloning andexpression vectors for use with bacterial, fungal, yeast, and mammaliancellular hosts are described, for example, in Pouwels et al. CloningVectors: A Laboratory Manual, Elsevier, N.Y., (1985). Cell-freetranslation systems could also be employed to produce TACE polypeptidesusing RNAs derived from DNA constructs disclosed herein.

[0039] Prokaryotes include gram negative or gram positive organisms, forexample, E. coli or Bacilli. Suitable prokaryotic host cells fortransformation include, for example, E. coli, Bacillus subtilis,Salmonella typhimurium, and various other species within the generaPseudomonas, Streptomyces, and Staphylococcus. In a prokaryotic hostcell, such as E. coli, a TACE polypeptide may include an N-terminalmethionine residue to facilitate expression of the recombinantpolypeptide in the prokaryotic host cell. The N-terminal Met may becleaved from the expressed recombinant TACE polypeptide.

[0040] Expression vectors for use in prokaryotic host cells generallycomprise one or more phenotypic selectable marker genes. A phenotypicselectable marker gene is, for example, a gene encoding a protein thatconfers antibiotic resistance or that supplies an autotrophicrequirement. Examples of useful expression vectors for prokaIyotic hostcells include those derived from conmercially available plasmids such asthe cloning vector pBR322 (ATCC 37017). pBR322 contains genes forampicillin and tetracycline resistance and thus provides simple meansfor identifying transformed cells. To construct en expression vectorusing pBR322, an appropriate promoter and a TACE DNA sequence areinserted into the pBR322 vector. Other commercially available vectorsinclude, for example, pKK223-3 (Phannacia Fine Chemicals, Uppsala,Sweden) and pGEM1 (Promega Biotec, Madison, Wis., USA).

[0041] Promoter sequences commonly used for recombinant prokaryotic hostcell expression vectors include Elactamase (penicillinase), lactosepromoter system (Chang et al., Nature 275:615, 1978; and Goeddel et al.,Nature 281:544, 1979), tryptophan (trp) promoter system (Goeddel et al.,Nucl. Acids Res. 8:4057, 1980; and EP-A-36776) and tac promoter(Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, p. 412, 1982). A particularly useful prokaryotic host cellexpression system employs a phage λ P_(L) promoter and a cI857tsthermolabile repressor sequence. Plasmid vectors available from theAmerican Type Culture Collection which incorporate derivatives of the λP_(L) promoter include plasrmid pHUB2 (resident in E. coli strain JMB9(ATCC 37092)) and pPLc28 (resident in E. coli RR1 (ATCC 53082)).

[0042] TACE polypeptides alternatively may be expressed in yeast hostcells, preferably from the Saccharomyces genus (e.g., S. cerevisiae).Other genera of yeast, such as Pichia, K. lactis or Kluyveromyces, mayalso be employed. Yeast vectors will often contain an origin ofreplication sequence from a 2μ yeast plasmid, an autonomouslyreplicating sequence (ARS), a promoter region, sequences forpolyadenylation, sequences for transcription termination, and aselectable marker gene. Suitable promoter sequences for yeast vectorsinclude, among others, promoters for metallothionein, 3-phosphoglyceratekinase (Hitzeman et al., J. Biol. Chem. 255:2073, 1980) or otherglycolytic enzymes (Hess et al., J. Adv. Enzyme Reg. 7:149, 1968; andHolland et al., Biochem. 17:4900, 1978), such as enolase,glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phosphoglucose isomerase, and glucokinase. Other suitable vectors andpromoters for use in yeast expression are further described in Hitzeman,EPA-73,657 or in Fleer et. al., Gene, 107:285-195 (1991); and van denBerg et. al., Bio/Technology, 8:135-139 (1990). Another alternative isthe glucose-repressible ADH2 promoter described by Russell et al. (J.Biol. Chem. 258:2674, 1982) and Beier et al. (Nature 300:724, 1982).Shuttle vectors replicable in both yeast and E. coli may be constructedby inserting DNA sequences from pBR322 for selection and replication inE. coli (Amp^(r) gene and origin of replication) into theabove-described yeast vectors.

[0043] The yeast α-factor leader sequence may be employed to directsecretion of a TACE polypeptide. The α-factor leader sequence is ofteninserted between the promoter sequence and the structural gene sequence.See, e.g., Kurjan et al., Cell 30:933, 1982; Bitter et al., Proc. Natl.Acad. Sci. USA 81:5330, 1984; U.S. Pat. No. 4,546,082; and EP 324,274.Other leader sequences suitable for facilitating secretion ofrecombinant polypeptides from yeast hosts are known to those of skill inthe art. A leader sequence may be modified near its 3′ end to containone or more restriction sites. This will facilitate fusion of the leadersequence to the structural gene.

[0044] Yeast transformation protocols are known to those of skill in theart. One such protocol is described by Hinnen et al., Proc. Natl. Acad.Sci. USA 75:1929, 1978. The Hinnen et al. protocol selects for Trp ⁺transformants in a selective medium, wherein the selective mediumconsists of 0.67% yeast nitrogen base, 0.5% casamino acids, 2% glucose,10 μg/ml adenine and 20 μg/ml uracil.

[0045] Yeast host cells transformed by vectors containing ADH2 promotersequence may be grown for inducing expression in a “rich” medium. Anexample of a rich medium is one consisting of 1% yeast extract, 2%peptone, and 1% glucose supplemented with 80 μg/ml adenine and 80 μg/mluracil. Derepression of the ADH2 promoter occurs when glucose isexhausted from the medium.

[0046] Mammalian or insect host cell culture systems could also beemployed to express recombinant TACE polypeptides. Baculovirus systemsfor production of heterologous proteins in insect cells are reviewed byLuckow and Summers, Bio/Technology 6:47 (1988). Established cell linesof mammalian origin also may be employed. Examples of suitable mammalianhost cell lines include the COS-7 line of monkey kidney cells (ATCC CRL1651) (Gluzman et al., Cell 23:175, 1981), L cells, C127 cells, 3T3cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells, HeLa cells, andBHK (ATCC CRL 10) cell lines, and the CV-1/EBNA-1 cell line derived fromthe African green monkey kidney cell line CVI (ATCC CCL 70) as describedby McMahan et aL (i EMBO J. 10: 2821, 1991).

[0047] Transcriptional and translational control sequences formamnmalian host cell expression vectors may be excised from viralgenomes. Commonly used promoter sequences and enhancer sequences arederived from Polyoma virus, Adenovirus 2, Simian Virus 40 (SV40), andhuman cytomegalovirus. DNA sequences derived from the SV40 viral genome,for example, SV40 origin, early and late promoter, enhancer, splice, andpolyadenylation sites may be used to provide other genetic elements forexpression of a structural gene sequence in a an host cell. Viral earlyand late promoters are particularly useful because both are easilyobtained from a viral genome as a fragment which may also contain aviral origin of replication (Fiers et al., Nature 273:113, 1978).Smaller or larger SV40 fragments may also be used, provided theapproximately 250 bp sequence extending from the Hind III site towardthe Bgl I site located in the SV40 viral origin of replication site isincluded.

[0048] Exemplary expression vectors for use in mammalian host cells canbe constructed as disclosed by Okayama and Berg (Mol. Cell. BioL 3:280,1983). A useful system for stable high level expression of mammaliancDNAs in C127 murine mammay epithelial cells can be constructedsubstantially as described by Cosman et al. (Mol. Immunol. 23:935,1986). A useful high expression vector, PMLSV N1/N4, described by Cosmanet al., Nature 312:768, 1984 has been deposited as ATCC 39890.Additional useful mammalian expression vectors are described inEP-A-0367566, and in U.S. patent application Ser. No. 07/701,415, filedMay 16, 1991, incorporated by reference herein. The vectors may bederived from retroviruses. In place of the native signal sequence, aheterologous signal sequence may be added, such as the signal sequencefor IL-7 described in U.S. Pat. No. 4,965,195; the signal sequence forIL-2 receptor described in Cosman et al., Nature 312:768 (1984); theIL-4 signal peptide described in EP 367,566; the type I IL-1 receptorsignal peptide described in U.S. Pat. No. 4,968,607; and the type IIIL-1 receptor signal peptide described in EP 460,846.

[0049] An isolated and purified TACE protein according to the inventionmay be produced by recombinant expression systems as described above orpurified from naturally occurring cells. TACE can be substantiallypurified, as indicated by a single protein band upon analysis bySDS-polyacrylamide gel electrophoresis (SDS-PAGE). One process forproducing TACE comprises culturing a host cell transformed with anexpression vector comprising a DNA sequence that encodes TACE underconditions sufficient to promote expression of TACE. TACE is thenrecovered from culture medium or cell extracts, depending upon theexpression system employed. As is known to the skilled artisan,procedures for purifying a recombinant protein will vary according tosuch factors as the type of host cells employed and whether or not therecombinant protein is secreted into the culture medium. For example,when expression systems that secrete the recombinant protein areemployed, the culture medium first may be concentrated using acommercially available protein concentration filter, for example, anAmicon or Millipore Pellicon ultrafiltration unit. Following theconcentration step, the concentrate can be applied to a purificationmatrix such as a gel filtration medium. Alternatively, an anion exchangeresin can be employed, for example, a matrix or substrate having pendantdiethylarninoethyl (DEAE) groups. The matrices can be acrylamide,agarose, dextran, cellulose or other types commonly employed in proteinpurification. Alternatively, a cation exchange step can be employed.Suitable cation exchangers include various insoluble matrices comprisingsulfopropyl or carboxymethyl groups. Sulfopropyl groups are preferred.Finally, one or more reversed-phase high performance liquidchromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,(e-g., silica gel having pendant methyl or other aliphatic groups) canbe employed to further purify TACE. Some or all of the foregoingpurification steps, in various combinations, are well known and can beemployed to provide an isolated and purified recombinant protein.

[0050] In addition to recombinantly producing TACE, TACE may be isolatedand purified from an activated monocytic cell line, THP-1. THP-1 cellstypically produce more TNF-α than do HL-60 cells, and are a preferedsource for TACE. Other sources for TACE may be used, and TACE may alsobe found in other types of cells that produce TNF-α. Once a source forTACE is identified, TACE may be isolated and purified by firstoptionally stimulating the source cells to produce TNF-α. Stimulationmay not be necessary, however, it can be done using techniques that arewell-known in the art. The cells are then harvested, washed, and plasmamembranes isolated according to conventional procedures. A particularlypreferred method of isolating the plasma membranes is method numberthree as described in Maeda et. al., Biochim. et. Biophys. Acta, 731:115(1983); except that dithiothreitol should not be included in this methodsince it was determined that dithiothreitol blocks TACE activity.Proteins from the cell membrane then can be solubilized by suspendingthe membrane preparation in a dilute solution of non-ionic detergent,followed by brief homogenization. Phospholipids then can be extractedusing conventional methods.

[0051] It is possible to utilize an affiity column comprising aTACE-binding protein to affinity-purify expressed TACE polypeptides.TACE polypeptides can be removed from an affinity column usingconventional techniques, e.g., in a high salt elution buffer and thendialyzed into a lower salt buffer for use or by changing pH or othercomponents depending on the affinity matrix utilized. Example 4describes a procedure for employing TACE of the invention to generatemonoclonal antibodies directed against TACE.

[0052] Recombinant protein produced in bacterial culture is usuallyisolated by initial disruption of the host cells, centrifugation,extraction from cell pellets if an insoluble polypeptide, or from thesupernatant fluid if a soluble polypeptide, followed by one or moreconcentration, salting-out, ion exchange, affinity purification or sizeexclusion chromatography steps. Finally, RP-HPLC can be employed forfinal purification steps. Microbial cells can be disrupted by anyconvenient method, including freeze-thaw cycling, sonication, mechanicaldisruption, or use of cell lysing agents.

[0053] Transformed yeast host cells are preferably employed to expressTACE as a secreted polypeptide in order to simplify purification.Secreted recombinant polypeptide from a yeast host cell fermentation canbe purified by methods analogous to those disclosed by Urdal et al. (J.Chromatog. 296:171, 1984). Urdal et al. describe two sequential,reversed-phase HPLC steps for purification of recombinant human IL-2 ona preparative HPLC column.

[0054] Antisense or sense oligonucleotides comprising a single-strandednucleic acid sequence (either RNA or DNA) capable of binding to a targetTACE mRNA sequence (fornring a duplex) or to the TACE sequence in thedouble-stranded DNA helix (forming a triple helix) can be made accordingto the invention. Antisense or sense oligonucleotides, according to thepresent invention, comprise a fragment of the coding region of TACEcDNA. Such a fragment generally comprises at least about 14 nucleotides,preferably from about 14 to about 30 nucleotides. The ability to createan antisense or a sense oligonucleotide, based upon a cDNA sequence fora given protein is described in, for example, Stein and Cohen, CancerRes. 48:2659, 1988 and van der Krol et al., BioTechniques 6:958, 1988.

[0055] Binding of antisense or sense oligonucleotides to target nucleicacid sequences results in the formation of complexes that blocktranslation (RNA) or transcription (DNA) by one of several means,including enhanced degradation of the duplexes, premature termination oftranscription or translation, or by other means. The antisenseoligonucleotides thus may be used to block expression of TACE proteins.Antisense or sense oligonucleotides further comprise oligo-nucleotideshaving modified sugar-phosphodiester backbones (or other sugar linkages,such as those described in WO91/06629) and wherein such sugar linkagesare resistant to endogenous nucleases. Such oligonucleotides withresistant sugar linkages are stable in vivo (i.e., capable of resistingenzymatic degradation) but retain sequence specificity to be able tobind to target nucleotide sequences. Other examples of sense orantisense oligonucleotides include those oligonucleotides which arecovalently linked to organic moieties, such as those described in WO90/10448, and other moieties that increases affinity of theoligonucleotide for a target nucleic acid sequence, such aspoly-(L-lysine). Further still, intercalating agents, such aseleipticine, and alkylating agents or metal complexes may be attached tosense or antisense oligonucleotides to modify binding specificities ofthe antisense or sense oliginucleotide for the target nucleotidesequence.

[0056] Antisense or sense oligonucleotides may be introduced into a cellcontaining the target nucleic acid sequence by any gene transfer method,including, for example, CaPO₄-mediated DNA transfection,electroporation, or by using gene transfer vectors such as Epstein-Barrvirus. Antisense or sense oligonucleotides are preferably introducedinto a cell containing the target nucleic acid sequence by insertion ofthe antisense or sense oligonucleotide into a suitable retroviralvector, then contacting the cell with the retrovirus vector containingthe inserted sequence, either in vivo or ex vivo. Suitable retroviralvectors include, but are not limited to, the murine retrovirus M-MULV,N2 (a retrovirus derived from M-MuLV), or or the double copy vectorsdesignated DCT5A, DCT5B and DCT5C (see PCT Application U.S. Pat. No.90/02656).

[0057] Sense or antisense oligonucleotides also may be introduced into acell containing the target nucleotide sequence by formation of aconjugate with a ligand binding molecule, as described in WO 91/04753.Suitable ligand binding molecules include, but are not limited to, cellsurface receptors, growth factors, other cytokines, or other ligandsthat bind to cell surface receptors. Preferably, conjugation of theligand binding molecule does not substantially interfere with theability of the ligand binding molecule to bind to its correspondingmolecule or receptor, or block entry of the sense or antisenseoligonucleotide or its conjugated version into the cell.

[0058] Alternatively, a sense or an antisense oligonucleotide may beintroduced into a cell containing the target nucleic acid sequence byformation of an oligonucleotide-lipid complex, as described in WO90/10448. The sense or antisense oligonucleotide-lipid complex ispreferably dissociated within the cell by an endogenous lipase.

[0059] Isolated and purified TACE or a fragment thereof, and inparticular, the extracellular domain of TACE, may also be useful itselfas a therapeutic agent in regulating the levels of certain cell surfaceproteins. In addition to TNF-α, other cytoidnes as well as cytokinereceptors and several adhesion proteins may be released from the cellsurface by TACE or related proteases. TACE or a fragment thereof, inparticular, the extracellular domain of TACE, may be administered tomodulate or remove cell surface cytokines, cytokine receptors andadhesion proteins involved in tumor cell growth, inflammation, orfertilization. When used as a therapeutic agent, TACE can be formulatedinto pharmaceutical compositions according to known methods. TACE can becombined in admixture, either as the sole active material or with otherknown active materials, with pharmaceutically suitable diluents (e.g.,Tris-HCl, acetate, phosphate), preservatives (e.g., Thimerosal, benzylalcohol, parabens), emulsifiers, solubilizers, adjuvants and/orcarriers. Suitable carriers and their formulations are described inRemington's Pharmaceutical Sciences, 16th ed. 1980, Mack Publishing Co.In addition, such compositions can contain TACE complexed withpolyethylene glycol (PEG), metal ions, or incorporated into polymericcompounds such as polyacetic acid, polyglycolic acid, hydrogels, etc.,or incorporated into liposomes, microemulsions, micelles, urilamellar ormultilamellar vesicles, erythrocyte ghosts or spheroblasts. Suchcompositions will influence the physical state, solubility, stability,rate of in vivo release, and rate of in vivo clearance of TACE.

[0060] TACE may be assayed using any of a variety of metalloproteaseassays known in the art. In general, TACE can be assayed through the useof a peptide substrate that represents the natural cleavage site ofTNF-α. For example, in order to detect the cleavage of a substrate byTACE, the substrate can be tagged with a fluorescent group on one sideof the cleavage site and with a fluorescence-quenching group on theopposite side of the cleavage site. Upon cleavage by TACE, quenching iseliminated thus providing a detectable signal. Alternatively, thesubstrate may be tagged with a colorimetric leaving group that morestrongly absorbs upon cleavage. Alternatively, the substrate may have athioester group synthesized into the cleavage site of the substrate sothat upon cleavage by TACE, the thiol group remains and can be easilydetected using conventional methods. A particularly preferred method ofdetecting TACE activity in a sample is described in Example 1, infra.Other methods of detecting TACE activity may be utilized withoutresorting to undue experimentation.

[0061] As further described in Example 1, infra, a quantitative assayfor TACE also may be used which assay involves incubating the peptidesubstrate, at about 1 mM, with TACE at 37° C. for a fixed period oftime; stopping the reaction by the addition of an acid or a metalchelator; and determining the extent of cleavage by HPLC analysis.

[0062] Within an aspect of the invention, TACE, and peptides based onthe amino acid sequence of TACE, may be utilized to prepare antibodiesthat specifically bind to TACE. A specific example of such antibodypreparation is described in Example 4 herein. The term “antibodies” ismeant to include polyclonal antibodies, monoclonal antibodies, fragmentsthereof such as F(ab')2, and Fab fragments, as well as any recombinantlyproduced binding partners. Antibodies are defined to be specificallybinding if they bind TACE with a K_(a) of greater than or equal to about10⁷ M⁻¹. Affinities of binding partners or antibodies can be readilydetermined using conventional techniques, for example those described byScatchard et al., Ann. N.Y. Acad. Sci., 51:660 (1949).

[0063] Polyclonal antibodies can be readily generated from a variety ofsources, for example, horses, cows, goats, sheep, dogs, chickens,rabbits, mice or rats, using procedures that are well-known in the art.In general, purified TACE, or a peptide based on the amino acid sequenceof TACE that is appropriately conjugated, is administered to the hostanimal typically through parenteral injection. The immunogenicity ofTACE may be enhanced through the use of an adjuvant, for example,Freund's complete or incomplete adjuvant. Following boosterimmunizations, small samples of serum are collected and tested forreactivity to TACE or the TACE peptides. Examples of various assaysuseful for such determination include those described in: Antibodies: ALaboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor LaboratoryPress, 1988; as well as procedures such as countercurrentimmuno-electrophoresis (CIEP), radio-immunoassay,radio-immunoprecipitation, enzyme-linked immuno-sorbent assays (FLISA),dot blot assays, and sandwich assays, see U.S. Pat. Nos. 4,376,110 and4,486,530.

[0064] Monoclonal antibodies may be readily prepared using well-knownprocedures, see for example, the procedures described in U.S. Pat. Nos.RE 32,011, 4,902,614, 4,543,439 and 4,411,993; Monoclonal Antibodies,Hybridomas: A New Dimension in Biological Analyses, Plenum Press,Kennett, McKeam, and Bechtol (eds.), 1980. Briefly, the host animals,such as mice are injected intraperitoneally at least once, andpreferably at least twice at about 3 week intervals with isolated andpurified TACE or conjugated TACE peptide, optionally in the presence ofadjuvant. Mouse sera are then assayed by conventional dot blot techniqueor antibody capture (ABC) to determine which animal is best to fuse.Approximately two to three weeks later, the mice are given anintravenous boost of TACE or conjugated TACE peptide. Mice are latersacrificed and spleen cells fused with commercially available myelomacells, such as Ag8.653 (ATCC), following established protocols. Briefly,the myeloma cells are washed several times in media and fused to mousespleen cells at a ratio of about thee spleen cells to one myeloma cell.The fusing agent can be any suitable agent used in the art, for example,polyethylene glycol (PEG). Fusion is plated out into plates containingmedia that allows for the selective growth of the fused cells. The fusedcells can then be allowed to grow for approximately eight days.Supernatants from resultant hybridomas are collected and added to aplate that is first coated with goat anti-mouse Ig. Following washes, alabel, such as, ¹²⁵I-TACE is added to each well followed by incubation.Positive wells can be subsequently detected by autoradiography. Positiveclones can be grown in bulk culture and supernatants are subsequentlypurified over a Protein A column (Pharmacia).

[0065] The monoclonal antibodies of the invention can be produced usingalternative techniques, such as those described by Alting-Mees et al.,“Monoclonal Antibody Expression Libraries: A Rapid Alternative toHybridomas”, Strategies in Molecular Biology 3:1-9 (1990) which isincorporated herein by reference. Similarly, binding partners can beconstructed using recombinant DNA techniques to incorporate the variableregions of a gene that encodes a specific binding antibody. Such atechnique is described in Larrick et al., Biotechnology, 7:394 (1989).

[0066] Other types of “antibodies” may be produced using the informationprovided herein in conjunction with the state of knowledge in the art.For example, humanized antibodies that are capable of specificallybinding TACE are also encompassed by the invention.

[0067] Once isolated and purified, the antibodies against TACE may beused to detect the presence of TACE in a sample using established assayprotocols. Further, the antibodies of the invention may be usedtherapeutically to bind to TACE and inhibit its activity in vivo.

[0068] The purified TACE according to the invention will facilitate thediscovery of inhibitors of TACE, and thus, inhibitors of excessiveTNF-α-release. The use of a purified TACE polypeptide in the screeningof potential inhibitors thereof is important and can virtually eliminatethe possibility of interfering reactions with contaminants. Such ascreening assay for detecting the TACE-inhibiting activity of a moleculewould typically involve mixing the potential inhibitor molecule with anappropriate substrate, incubating TACE that is at least substantiallypurified with the mixture, and determining the extent of substratecleavage as, for example, described above. While various appropriatesubstrates may be designed for use in the assay, preferably, a peptidylsubstrate is used, and which substrate comprises the amino acid sequenceLeu-Ala-Gln-Ala-Val-Arg-Ser-Ser (SEQ ID NO:5).

[0069] In addition, TACE polypeptides can also be used forstructure-based design of TACE-inhibitors. Such structure-based designis also known as “rational drug design.” The TACE polypeptides can bethree-dimensionally analyzed by, for example, X-ray crystallography,nuclear magnetic resonance or homology modeling, all of which arewell-known methods. The use of TACE structural information in molecularmodeling software systems to assist in inhibitor design andinhibitor-TACE interaction is also encompassed by the invention. Suchcomputer-assisted modeling and drug design may utilize information suchas chemical conformational analysis, electrostatic potential of themolecules, protein folding, etc. For example, most of the design ofclass-specific inhibitors of metalloproteases has focused on attempts tochelate or bind the catalytic zinc atom. Synthetic inhibitors areusually designed to contain a negatively-charged moiety to which isattached a series of other groups designed to fit the specificitypockets of the particular protease. A particular method of the inventioncomprises analyzing the three dimensional structure of TACE for likelybinding sites of substrates, synthesizing a new molecule thatincorporates a predictive reactive site, and assaying the new moleculeas described above.

[0070] The following Examples provide an illustration of embodiments ofthe invention and should not be construed to limit the scope of theinvention which is set forth in the appended claims. In the followingExamples, all methods described are conventional unless otherwisespecified.

EXAMPLE 1 Purification of the TNF-α Converting Enzyme

[0071] This Example describes a method for purifying TACE. The TACE wasisolated and purified from the membranes of the human monocytic cellline, THP-1, (ATCC no. TIB 202) that had been stimulated to produceTNF-α. THP-1 cells were chosen because they produce more TNF-α thanHL-60 cells, a more commonly used human monocytic cell line.Approximately 120 billion cells were stimulated using the procedurepreviously described by Kronheim et al., Arch. Biochemn. Biophys.269:698 (1992), incorporated herein by reference. Two hours afterstimulation, the cells were harvested by centrifugation. The harvestedcells were washed at least twice with Hanks balanced salt solution, andplasma membranes were isolated according to method number three asdescribed by Maeda et. al., Biochim. et. Biophys. Acta, 731:115 (1983),except that dithiothreitol was not used, utilizing 1.25 ml ofhomogenization buffer per ml of cell pellet. It was determined that thestandard procedure of Maeda et al., Id., utilizing dithiothreitol,failed to yield compounds having TACE activity (an assay for TACEactivity is described below). Proteins were then solubilized byresuspending the membrane preparation in a solution of 1%octylglucoside, 10 mM Tris-HCl (pH 8), 1 mM MgCl₂ and 30 mM NaCl andbriefly homogenizing with a Briiiran Homogenizer (twice, five secondseach time). Phospholipids were then extracted by adding four volumes ofice-cold (0° C.) acetone; after a thirty-minute incubation at 4° C., theacetone-extracted material was centrifuged at 1500 rpm for 10 minutes ina H1000B rotor.

[0072] Chromatography

[0073] The pelleted material was dissolved in 450 ml of Buffer A (3ufferA comprises 10 mM Tris-HCl (pH 7.5) and 1% octylglucoside (weight tovolume percent)) and applied to a 120 mnl column of DEAE-Sepharosefast-flow (Pharmacia) at 4 ml per minute. The column then was washedwith 360 ml of Buffer A at 6 ml per minute, and protein was then elutedwith an increasing gradient of NaCl (0-0.3 M) in Buffer A applied at 6ml per minute over a period of 40 minutes. TACE was eluted with a NaClconcentration of about 50 to about 150 mM.

[0074] TACE was originally detected at this point by its ability tocleave recombinant 26 kD TNF-α fused to the “flag” (T. P. Hopp, et al.,Bio Technology, 6:1204 (1988) sequence of 8 amino acids at theamino-terminus. The gene encoding human TNF-α was spliced to DNAencoding the flag sequence, and this construct was placed in the pPL3vector (C. Maliszewski et al., Molec. Immnunol., 25:429 (1987). Theprotein was then expressed in a protease-deficient strain of E. coli (R.T. Libby et al., DNA, 6:221 (1987) which was found necessary to preventdegradation of the precursor by the bacteria After removal of growthmedium, the bacteria were resuspended in 30 mM Tris-HCl (pH 8), 5 mMEDTA, and the suspension was sonicated for about 30 seconds. Thematerial was then centrifuged at 20,000 rpm in an SS34 rotor for 30minutes, the supernatant fraction was discarded, and the pellet wasresuspended with 8 M urea in 10 mM Tris-HCl (pH 8). The material washomogenized with 25 strokes in a dounce homogenizer and then centrifugedat 20,000 rpm in an SS34 rotor for 30 minutes. The supernatant fraction,which contained the precursor TNF-α, was then dialyzed four timesagainst 10 mM Tris-HCl (pH 8).

[0075] This material was incubated at 37° C. for at least 4 hours withthe TACE eluted from the DEAE-Sepharose, that had been treated with 1 mMN-methoxysuccinyl-Ala-Ala-Val-chloro-methylketone, 10 μg/ml leupeptin,and 1 mg/ml α1-protease inhibitor, all of which are commerciallyavailable. The N-terminus of the resulting 17 kD product was found to bethat of authentic TNF-α. After the initial identification of TACE inthis way, it was found that the enzyme also cleaves an 8-residue peptiderepresenting the segmentLeu⁷³-Ala⁷⁴-Gln⁷⁵-Ala⁷⁶-↓-Val⁷⁷-Arg⁷⁸-Ser⁷⁹-Ser⁸⁰ (SEQ ID NO;5) ofTNF-α. Wherein the (↓) illustrates the cleavage site. Based on thisobservation, a quantitative assay was established: the peptide, at 1 mM,was incubated with the enzyme at 37° C. for a fixed period of time, inthe presence of 0.1 mM dichloroisocoumarin, 1 mMmethoxysuccinyl-Ala-Ala-ProVal-chloromethyl-ketone, 10 μg/ml leupeptin,10 μM bestatin, and 1 mg/ml α1-protease inhibitor (Sigma), all of whichare commercially available. The reaction was then stopped by theaddition of acid or a metal chelator. The extent of cleavage of thispeptide, reflecting the amount of TACE present, was determined byapplying the mixture to a Vydac C18 column and eluting with a gradientof 0 to 30% acetonitrile over a period of 15 minutes.

[0076] Material that eluted from the DEAE column with 0.05-0.25 M NaClhad about a 4-fold higher specific activity than the starting material.The eluted material was sonicated and then shaken with wheat germagglutinin-agarose (Vector Laboratories) for two hours at 4° C. Prior touse, the wheat germ agglutinin-agarose was washed with 5 column volumesof Buffer B (Buffer B comprises 10 mM Tris-HCl (pH 7.5), 0.15 M NaCI,0.1 mM MnCl₂, 0.1 mM CaCl₂, 1% octylglucoside and 10% glycerol); 1 ml ofthis resin was used for every 2 mg of protein in the sample, asdetermined by the BCA protein assay (Pierce). After two hours, the resinwas washed with 7 volumes of Buffer B, and material was then eluted with5 column volumes of Buffer B plus 0.3 M acetylglucosamine (Sigma), with30 minute intervals between the application of each column volume.

[0077] Eluted fractions containing TACE activity had about a ten-foldhigher specific activity than the starting material. These fractionswere concentrated to about 5 ml with Centriprep-30 concentrators(Amicon) and then diluted three-fold with Buffer C (Buffer C comprises10 mM Tris-HCl (pH 8), 1% octylglucoside and 10% glycerol). The dilutedmaterial was sonicated (three 10-second bursts) and then loaded onto aMonoQ HR 5/5 column (Pharmacia) at 0.5 ml per minute. The column wasthen washed with 10 ml of Buffer C at 0.5 ml per minute, and materialwas eluted with a 0 to 0.25 M NaCl gradient in Buffer C at 0.5 ml perminute over a period of 30 minutes. TACE activity (detected at thisstage and subsequently by incubation with the previously describedpeptide substrate in the absence of protease inhibitors) eluted withabout 0. 15M NaCl.

[0078] The NaCl concentration in the MonoQ fractions containing activitywas reduced by at least ten-fold by diluting the material into Buffer C,and the material was then applied to a column of hydroxyapatite(American International Chemical, ceramic hydroxyapatite HS40) at therate of 0.5 ml per minute. After washing with three column volumes ofBuffer C, protein was eluted with a 0 to 50 mM gradient of sodiumphosphate at 1 ml per minute over a period of 30 minutes. TACE elutedwith about 15 mM sodium phosphate.

[0079] The TACE eluted from the hydroxyapatite column was thenconcentrated to about 100 μl with Centricon-50 concentrators (Amicon)and applied to a Bio-Rad SEC-400 sizing column (30 cm). Protein waseluted with Buffer C run through the column at 0.5 ml per minute; TACEeluted at about 28 minutes.

[0080] The TACE eluted from the sizing column was- diluted three-foldinto Buffer D (Buffer D comprises 20 mM MES (pH 6), 1% octyglucoside and10% glycerol) and applied to a 1 ml column of Red 120-agrose (Sigrna) at0.25 ml per minute. After the column was washed with 10 ml Buffer D,protein was eluted with a 0 to 1 M NaCl gradient in Buffer D at 0.25 mlper minute over a period of 60 minutes. TACE eluted with 0.2 to 0.3 MNaCl. Five percent of each eluted fraction was run on aSDS-polyacrylamide gel (10%), and silver staining showed that thepredominant protein in the fractions with activity ran approximatelymidway between the 66 and 97 kD markers (Novex) on the gel, atapproximately 80 kD.

[0081] Trifluoroacetic acid (IfFA) was added to 0.2% (volume-to-volumnepercentage) to a pool of the fractions containing the approximately 80kD protein, and the mixture was then pumped onto a 2.1×5 cm C4 column atapproximately 100 μl per minute using a Shimadzu LC-10AD. Protein waseluted with a 0 to 100% gradient of acetonitrule in 0.1% TFA at 100 μlper minute over a period of 100 minutes. One minute fractions werecollected and 5 to 10% of each fraction was run on a NovexSDS-polyacrylamide gel (10%). Fractions that eluted with about 70%acetonitrile and that contained a protein of approximately 80 kD werepooled and evaporated to dryness.

[0082] Generation of peptides and sequencing

[0083] This pool of fractions then was dissolved in 200 μl of 50 mMTris-HCl (pH 8), 1 mM EDTA, and an amount of endo-LYS-C (Promega) equalto about {fraction (1/50)} of the amount of protein in the sample wasadded. The material was incubated at 37° C. overnight, and then a freshaliquot of the same amount of endo-LYS-C was added for an additional 3hours at 37° C.

[0084] The resulting peptides were separated by applying the material toa capillary C18 column at 20 μl per minute and eluting with an ascendinggradient of acetonluile (0.5% per minute) in 0.1% TFA over a period of200 minutes. Peptides were sequenced with an ABI 476 or an ABI 494automated sequencer.

EXAMPLE 2 Preparation of Isolated and Purified TACE

[0085] This Example describes a method for further purifying thepurified TACE as was obtained using the procedures described above.Purified TACE obtained from the THP-1 cells may contain small amounts ofhuman lysosomal 85 kD sialoglycoprotein (Biochem. Biophys. Res. Commun.184:604-611 (1992) and human lysosomal alpha-mannosidase (Biochem.Biophys. Res. Comm. 200:239-245 (1994) that can be removed usingstandard innmunoadsorbant procedures, as described in, for example,Robert K. Scopes, Protein Purification-Principles and Practice(Springer-Verlag, 2nd edit), pp. 167-172. Using the procedures describedin this Example 2, isolated and purified TACE can be obtained.

EXAMPLE 3 Cloning of Human TACE

[0086] This example describes a procedure for isolating a DNA sequenceencoding human TACE. A random primed cDNA library was generated from thecommercially available cell line THP-1 (Amersham) using conventionalmethods. Polymerase chain reaction (PCR) (Mullis and Faloona, Meth.Enzymol. 155:335-350, 1987) amplifications were performed using thefollowing primers:

[0087] Primer (1): 5′-AARTAYGTNATGTAYCC-3′ SEQ ID NO:6

[0088] Primer (2): 5′-CCRCARTCRCAYTCYTC-3′ SEQ ID NO:7

[0089] Primer (1) is based on the first five amino acids of Peptide (2)with the addition of a triplet coding for lysine at the 5′ end. Primer(2) is antisense to a conserved amino acid sequenceGlu-Glu-Cys-Asp-Cys-Gly (EECDCG) SEQ ID NO:8, which is found in ahomologous metalloprotease, bovine reprolysin 1 (GenBank Accession#Z2196 1).

[0090] Single stranded cDNA was amplified using the mixedoligonucleotides described above under standard PCR conditions. The PCRreaction products were fractionated by gel electrophoresis and DNA bandsof approximately 180 bp were isolated and subcloned into commerciallyavailable pBLUESCRIPT. Sequencing revealed a clone that contained anucleotide sequence that codes for the amino acidsIle-Ala-Val-Ser-Gly-Asp-His-Glu-Asn-Asn-Lys (SEQ ID NO:9) and anucleotide sequence that codes for amino acids Glu-Glu-Cys-Asp-Cys-Gly(EECDCG) (SEQ ID NO:8). This clone was termed the “30CD clone.” The 30CDclone was sequenced and primers were generated based on this sequence.The primers then were used to detect TACE cDNA in phage library madefrom human KB cells. This library was screened under conventionalconditions using a probe based on the 30CD sequence. Positivehybridizing plaques were isolated and DNA fragments of these clones weresequenced. Sequencing provided a full length cDNA of human TACE which isshown in SEQ ID NO:1. Human TACE was found to be a type I transmembraneprotein of 824 amino acids, including a N-terminal 17 amino acid signalpeptide. The signal peptide is followed by an extracellular domain of654 amino acids, a 23 amino acid transmembrane domain and a 130 aminoacid cytoplasmic domain. An alternate spliced variant was cloned andsequenced and found to contain the same amino acid sequence as TACE,except that a 50 bp fragment is deleted at the 5′ end of the cytoplasmicdomain, thus shifting the reading frame to encode a six amino acidcytoplasmic domain. The amino acid sequence of this variant is shown inSEQ ID NO:4, with the cDNA shown in SEQ ID NO:3.

EXAMPLE4 Preparation of Antibodies Against TACE

[0091] This Example describes a method for generating monoclonalantibodies against TACE. Balb/c mice are injected intraperitoneally ontwo occasions at 3 week intervals with 10 ug of isolated and purifiedTACE of Example 1 or peptides based on the amino acid sequence of TACEin the presence of RIBI adjuvant (RIBI Corp., Hamilton, Mo.). Mouse seraare then assayed by conventional dot blot technique or antibody capture(ABC) to determine which animal is best to fuse. Three weeks later, miceare given an intravenous boost of 3 ug of human TACE, or TACE peptide,suspended in sterile PBS. Three days later, mice are sacrificed andspleen cells fused with Ag8.653 myeloma cells (ATCC) followingestablished protocols. Briefly, Ag8.653 cells are washed several timesin serum-free media and fused to mouse spleen cells at a ratio of threespleen cells to one myeloma cell. The fusing agent is 50% PEG: 10% DMSO(Sigma). Fusion is plated out into twenty 96-well flat bottom plates(Corning) containing HAT supplemented DMEM media and allowed to grow foreight days. Supematants from resultant hybridomas are collected andadded to a 96-well plate for 60 minutes that is first coated with goatanti-mouse Ig. Following washes, ¹²⁵I-TACE is added to each well,incubated for 60 minutes at room temperature, and washed four times.Positive wells can be subsequently detected by autoradiography at −70°C. using Kodak X-Omat S film. Positive clones can be grown in bulkculture and supernatants are subsequently purified over a Protein Acolumn (Pharmacia).

1 9 1 2475 DNA Homo sapiens CDS (1)..(2472) 1 atg agg cag tct ctc ctattc ctg acc agc gtg gtt cct ttc gtg ctg 48 Met Arg Gln Ser Leu Leu PheLeu Thr Ser Val Val Pro Phe Val Leu 1 5 10 15 gcg ccg cga cct ccg gatgac ccg ggc ttc ggc ccc cac cag aga ctc 96 Ala Pro Arg Pro Pro Asp AspPro Gly Phe Gly Pro His Gln Arg Leu 20 25 30 gag aag ctt gat tct ttg ctctca gac tac gat att ctc tct tta tct 144 Glu Lys Leu Asp Ser Leu Leu SerAsp Tyr Asp Ile Leu Ser Leu Ser 35 40 45 aat atc cag cag cat tcg gta agaaaa aga gat cta cag act tca aca 192 Asn Ile Gln Gln His Ser Val Arg LysArg Asp Leu Gln Thr Ser Thr 50 55 60 cat gta gaa aca cta cta act ttt tcagct ttg aaa agg cat ttt aaa 240 His Val Glu Thr Leu Leu Thr Phe Ser AlaLeu Lys Arg His Phe Lys 65 70 75 80 tta tac ctg aca tca agt act gaa cgtttt tca caa aat ttc aag gtc 288 Leu Tyr Leu Thr Ser Ser Thr Glu Arg PheSer Gln Asn Phe Lys Val 85 90 95 gtg gtg gtg gat ggt aaa aac gaa agc gagtac act gta aaa tgg cag 336 Val Val Val Asp Gly Lys Asn Glu Ser Glu TyrThr Val Lys Trp Gln 100 105 110 gac ttc ttc act gga cac gtg gtt ggt gagcct gac tct agg gtt cta 384 Asp Phe Phe Thr Gly His Val Val Gly Glu ProAsp Ser Arg Val Leu 115 120 125 gcc cac ata aga gat gat gat gtt ata atcaga atc aac aca gat ggg 432 Ala His Ile Arg Asp Asp Asp Val Ile Ile ArgIle Asn Thr Asp Gly 130 135 140 gcc gaa tat aac ata gag cca ctt tgg agattt gtt aat gat acc aaa 480 Ala Glu Tyr Asn Ile Glu Pro Leu Trp Arg PheVal Asn Asp Thr Lys 145 150 155 160 gac aaa aga atg tta gtt tat aaa tctgaa gat atc aag aat gtt tca 528 Asp Lys Arg Met Leu Val Tyr Lys Ser GluAsp Ile Lys Asn Val Ser 165 170 175 cgt ttg cag tct cca aaa gtg tgt ggttat tta aaa gtg gat aat gaa 576 Arg Leu Gln Ser Pro Lys Val Cys Gly TyrLeu Lys Val Asp Asn Glu 180 185 190 gag ttg ctc cca aaa ggg tta gta gacaga gaa cca cct gaa gag ctt 624 Glu Leu Leu Pro Lys Gly Leu Val Asp ArgGlu Pro Pro Glu Glu Leu 195 200 205 gtt cat cga gtg aaa aga aga gct gaccca gat ccc atg aag aac acg 672 Val His Arg Val Lys Arg Arg Ala Asp ProAsp Pro Met Lys Asn Thr 210 215 220 tgt aaa tta ttg gtg gta gca gat catcgc ttc tac aga tac atg ggc 720 Cys Lys Leu Leu Val Val Ala Asp His ArgPhe Tyr Arg Tyr Met Gly 225 230 235 240 aga ggg gaa gag agt aca act acaaat tac tta ata gag cta att gac 768 Arg Gly Glu Glu Ser Thr Thr Thr AsnTyr Leu Ile Glu Leu Ile Asp 245 250 255 aga gtt gat gac atc tat cgg aacact tca tgg gat aat gca ggt ttt 816 Arg Val Asp Asp Ile Tyr Arg Asn ThrSer Trp Asp Asn Ala Gly Phe 260 265 270 aaa ggc tat gga ata cag ata gagcag att cgc att ctc aag tct cca 864 Lys Gly Tyr Gly Ile Gln Ile Glu GlnIle Arg Ile Leu Lys Ser Pro 275 280 285 caa gag gta aaa cct ggt gaa aagcac tac aac atg gca aaa agt tac 912 Gln Glu Val Lys Pro Gly Glu Lys HisTyr Asn Met Ala Lys Ser Tyr 290 295 300 cca aat gaa gaa aag gat gct tgggat gtg aag atg ttg cta gag caa 960 Pro Asn Glu Glu Lys Asp Ala Trp AspVal Lys Met Leu Leu Glu Gln 305 310 315 320 ttt agc ttt gat ata gct gaggaa gca tct aaa gtt tgc ttg gca cac 1008 Phe Ser Phe Asp Ile Ala Glu GluAla Ser Lys Val Cys Leu Ala His 325 330 335 ctt ttc aca tac caa gat tttgat atg gga act ctt gga tta gct tat 1056 Leu Phe Thr Tyr Gln Asp Phe AspMet Gly Thr Leu Gly Leu Ala Tyr 340 345 350 gtt ggc tct ccc aga gca aacagc cat gga ggt gtt tgt cca aag gct 1104 Val Gly Ser Pro Arg Ala Asn SerHis Gly Gly Val Cys Pro Lys Ala 355 360 365 tat tat agc cca gtt ggg aagaaa aat atc tat ttg aat agt ggt ttg 1152 Tyr Tyr Ser Pro Val Gly Lys LysAsn Ile Tyr Leu Asn Ser Gly Leu 370 375 380 acg agc aca aag aat tat ggtaaa acc atc ctt aca aag gaa gct gac 1200 Thr Ser Thr Lys Asn Tyr Gly LysThr Ile Leu Thr Lys Glu Ala Asp 385 390 395 400 ctg gtt aca act cat gaattg gga cat aat ttt gga gca gaa cat gat 1248 Leu Val Thr Thr His Glu LeuGly His Asn Phe Gly Ala Glu His Asp 405 410 415 ccg gat ggt cta gca gaatgt gcc ccg aat gag gac cag gga ggg aaa 1296 Pro Asp Gly Leu Ala Glu CysAla Pro Asn Glu Asp Gln Gly Gly Lys 420 425 430 tat gtc atg tat ccc atagct gtg agt ggc gat cac gag aac aat aag 1344 Tyr Val Met Tyr Pro Ile AlaVal Ser Gly Asp His Glu Asn Asn Lys 435 440 445 atg ttt tca aac tgc agtaaa caa tca atc tat aag acc att gaa agt 1392 Met Phe Ser Asn Cys Ser LysGln Ser Ile Tyr Lys Thr Ile Glu Ser 450 455 460 aag gcc cag gag tgt tttcaa gaa cgc agc aat aaa gtt tgt ggg aac 1440 Lys Ala Gln Glu Cys Phe GlnGlu Arg Ser Asn Lys Val Cys Gly Asn 465 470 475 480 tcg agg gtg gat gaagga gaa gag tgt gat cct ggc atc atg tat ctg 1488 Ser Arg Val Asp Glu GlyGlu Glu Cys Asp Pro Gly Ile Met Tyr Leu 485 490 495 aac aac gac acc tgctgc aac agc gac tgc acg ttg aag gaa ggt gtc 1536 Asn Asn Asp Thr Cys CysAsn Ser Asp Cys Thr Leu Lys Glu Gly Val 500 505 510 cag tgc agt gac aggaac agt cct tgc tgt aaa aac tgt cag ttt gag 1584 Gln Cys Ser Asp Arg AsnSer Pro Cys Cys Lys Asn Cys Gln Phe Glu 515 520 525 act gcc cag aag aagtgc cag gag gcg att aat gct act tgc aaa ggc 1632 Thr Ala Gln Lys Lys CysGln Glu Ala Ile Asn Ala Thr Cys Lys Gly 530 535 540 gtg tcc tac tgc acaggt aat agc agt gag tgc ccg cct cca gga aat 1680 Val Ser Tyr Cys Thr GlyAsn Ser Ser Glu Cys Pro Pro Pro Gly Asn 545 550 555 560 gct gaa gat gacact gtt tgc ttg gat ctt ggc aag tgt aag gat ggg 1728 Ala Glu Asp Asp ThrVal Cys Leu Asp Leu Gly Lys Cys Lys Asp Gly 565 570 575 aaa tgc atc cctttc tgc gag agg gaa cag cag ctg gag tcc tgt gca 1776 Lys Cys Ile Pro PheCys Glu Arg Glu Gln Gln Leu Glu Ser Cys Ala 580 585 590 tgt aat gaa actgac aac tcc tgc aag gtg tgc tgc agg gac ctt tcc 1824 Cys Asn Glu Thr AspAsn Ser Cys Lys Val Cys Cys Arg Asp Leu Ser 595 600 605 ggc cgc tgt gtgccc tat gtc gat gct gaa caa aag aac tta ttt ttg 1872 Gly Arg Cys Val ProTyr Val Asp Ala Glu Gln Lys Asn Leu Phe Leu 610 615 620 agg aaa gga aagccc tgt aca gta gga ttt tgt gac atg aat ggc aaa 1920 Arg Lys Gly Lys ProCys Thr Val Gly Phe Cys Asp Met Asn Gly Lys 625 630 635 640 tgt gag aaacga gta cag gat gta att gaa cga ttt tgg gat ttc att 1968 Cys Glu Lys ArgVal Gln Asp Val Ile Glu Arg Phe Trp Asp Phe Ile 645 650 655 gac cag ctgagc atc aat act ttt gga aag ttt tta gca gac aac atc 2016 Asp Gln Leu SerIle Asn Thr Phe Gly Lys Phe Leu Ala Asp Asn Ile 660 665 670 gtt ggg tctgtc ctg gtt ttc tcc ttg ata ttt tgg att cct ttc agc 2064 Val Gly Ser ValLeu Val Phe Ser Leu Ile Phe Trp Ile Pro Phe Ser 675 680 685 att ctt gtccat tgt gtg gat aag aaa ttg gat aaa cag tat gaa tct 2112 Ile Leu Val HisCys Val Asp Lys Lys Leu Asp Lys Gln Tyr Glu Ser 690 695 700 ctg tct ctgttt cac ccc agt aac gtc gaa atg ctg agc agc atg gat 2160 Leu Ser Leu PheHis Pro Ser Asn Val Glu Met Leu Ser Ser Met Asp 705 710 715 720 tct gcatcg gtt cgc att atc aaa ccc ttt cct gcg ccc cag act cca 2208 Ser Ala SerVal Arg Ile Ile Lys Pro Phe Pro Ala Pro Gln Thr Pro 725 730 735 ggc cgcctg cag cct gcc cct gtg atc cct tcg gcg cca gca gct cca 2256 Gly Arg LeuGln Pro Ala Pro Val Ile Pro Ser Ala Pro Ala Ala Pro 740 745 750 aaa ctggac cac cag aga atg gac acc atc cag gaa gac ccc agc aca 2304 Lys Leu AspHis Gln Arg Met Asp Thr Ile Gln Glu Asp Pro Ser Thr 755 760 765 gac tcacat atg gac gag gat ggg ttt gag aag gac ccc ttc cca aat 2352 Asp Ser HisMet Asp Glu Asp Gly Phe Glu Lys Asp Pro Phe Pro Asn 770 775 780 agc agcaca gct gcc aag tca ttt gag gat ctc acg gac cat ccg gtc 2400 Ser Ser ThrAla Ala Lys Ser Phe Glu Asp Leu Thr Asp His Pro Val 785 790 795 800 accaga agt gaa aag gct gcc tcc ttt aaa ctg cag cgt cag aat cgt 2448 Thr ArgSer Glu Lys Ala Ala Ser Phe Lys Leu Gln Arg Gln Asn Arg 805 810 815 gttgac agc aaa gaa aca gag tgc taa 2475 Val Asp Ser Lys Glu Thr Glu Cys 8202 824 PRT Homo sapiens 2 Met Arg Gln Ser Leu Leu Phe Leu Thr Ser Val ValPro Phe Val Leu 1 5 10 15 Ala Pro Arg Pro Pro Asp Asp Pro Gly Phe GlyPro His Gln Arg Leu 20 25 30 Glu Lys Leu Asp Ser Leu Leu Ser Asp Tyr AspIle Leu Ser Leu Ser 35 40 45 Asn Ile Gln Gln His Ser Val Arg Lys Arg AspLeu Gln Thr Ser Thr 50 55 60 His Val Glu Thr Leu Leu Thr Phe Ser Ala LeuLys Arg His Phe Lys 65 70 75 80 Leu Tyr Leu Thr Ser Ser Thr Glu Arg PheSer Gln Asn Phe Lys Val 85 90 95 Val Val Val Asp Gly Lys Asn Glu Ser GluTyr Thr Val Lys Trp Gln 100 105 110 Asp Phe Phe Thr Gly His Val Val GlyGlu Pro Asp Ser Arg Val Leu 115 120 125 Ala His Ile Arg Asp Asp Asp ValIle Ile Arg Ile Asn Thr Asp Gly 130 135 140 Ala Glu Tyr Asn Ile Glu ProLeu Trp Arg Phe Val Asn Asp Thr Lys 145 150 155 160 Asp Lys Arg Met LeuVal Tyr Lys Ser Glu Asp Ile Lys Asn Val Ser 165 170 175 Arg Leu Gln SerPro Lys Val Cys Gly Tyr Leu Lys Val Asp Asn Glu 180 185 190 Glu Leu LeuPro Lys Gly Leu Val Asp Arg Glu Pro Pro Glu Glu Leu 195 200 205 Val HisArg Val Lys Arg Arg Ala Asp Pro Asp Pro Met Lys Asn Thr 210 215 220 CysLys Leu Leu Val Val Ala Asp His Arg Phe Tyr Arg Tyr Met Gly 225 230 235240 Arg Gly Glu Glu Ser Thr Thr Thr Asn Tyr Leu Ile Glu Leu Ile Asp 245250 255 Arg Val Asp Asp Ile Tyr Arg Asn Thr Ser Trp Asp Asn Ala Gly Phe260 265 270 Lys Gly Tyr Gly Ile Gln Ile Glu Gln Ile Arg Ile Leu Lys SerPro 275 280 285 Gln Glu Val Lys Pro Gly Glu Lys His Tyr Asn Met Ala LysSer Tyr 290 295 300 Pro Asn Glu Glu Lys Asp Ala Trp Asp Val Lys Met LeuLeu Glu Gln 305 310 315 320 Phe Ser Phe Asp Ile Ala Glu Glu Ala Ser LysVal Cys Leu Ala His 325 330 335 Leu Phe Thr Tyr Gln Asp Phe Asp Met GlyThr Leu Gly Leu Ala Tyr 340 345 350 Val Gly Ser Pro Arg Ala Asn Ser HisGly Gly Val Cys Pro Lys Ala 355 360 365 Tyr Tyr Ser Pro Val Gly Lys LysAsn Ile Tyr Leu Asn Ser Gly Leu 370 375 380 Thr Ser Thr Lys Asn Tyr GlyLys Thr Ile Leu Thr Lys Glu Ala Asp 385 390 395 400 Leu Val Thr Thr HisGlu Leu Gly His Asn Phe Gly Ala Glu His Asp 405 410 415 Pro Asp Gly LeuAla Glu Cys Ala Pro Asn Glu Asp Gln Gly Gly Lys 420 425 430 Tyr Val MetTyr Pro Ile Ala Val Ser Gly Asp His Glu Asn Asn Lys 435 440 445 Met PheSer Asn Cys Ser Lys Gln Ser Ile Tyr Lys Thr Ile Glu Ser 450 455 460 LysAla Gln Glu Cys Phe Gln Glu Arg Ser Asn Lys Val Cys Gly Asn 465 470 475480 Ser Arg Val Asp Glu Gly Glu Glu Cys Asp Pro Gly Ile Met Tyr Leu 485490 495 Asn Asn Asp Thr Cys Cys Asn Ser Asp Cys Thr Leu Lys Glu Gly Val500 505 510 Gln Cys Ser Asp Arg Asn Ser Pro Cys Cys Lys Asn Cys Gln PheGlu 515 520 525 Thr Ala Gln Lys Lys Cys Gln Glu Ala Ile Asn Ala Thr CysLys Gly 530 535 540 Val Ser Tyr Cys Thr Gly Asn Ser Ser Glu Cys Pro ProPro Gly Asn 545 550 555 560 Ala Glu Asp Asp Thr Val Cys Leu Asp Leu GlyLys Cys Lys Asp Gly 565 570 575 Lys Cys Ile Pro Phe Cys Glu Arg Glu GlnGln Leu Glu Ser Cys Ala 580 585 590 Cys Asn Glu Thr Asp Asn Ser Cys LysVal Cys Cys Arg Asp Leu Ser 595 600 605 Gly Arg Cys Val Pro Tyr Val AspAla Glu Gln Lys Asn Leu Phe Leu 610 615 620 Arg Lys Gly Lys Pro Cys ThrVal Gly Phe Cys Asp Met Asn Gly Lys 625 630 635 640 Cys Glu Lys Arg ValGln Asp Val Ile Glu Arg Phe Trp Asp Phe Ile 645 650 655 Asp Gln Leu SerIle Asn Thr Phe Gly Lys Phe Leu Ala Asp Asn Ile 660 665 670 Val Gly SerVal Leu Val Phe Ser Leu Ile Phe Trp Ile Pro Phe Ser 675 680 685 Ile LeuVal His Cys Val Asp Lys Lys Leu Asp Lys Gln Tyr Glu Ser 690 695 700 LeuSer Leu Phe His Pro Ser Asn Val Glu Met Leu Ser Ser Met Asp 705 710 715720 Ser Ala Ser Val Arg Ile Ile Lys Pro Phe Pro Ala Pro Gln Thr Pro 725730 735 Gly Arg Leu Gln Pro Ala Pro Val Ile Pro Ser Ala Pro Ala Ala Pro740 745 750 Lys Leu Asp His Gln Arg Met Asp Thr Ile Gln Glu Asp Pro SerThr 755 760 765 Asp Ser His Met Asp Glu Asp Gly Phe Glu Lys Asp Pro PhePro Asn 770 775 780 Ser Ser Thr Ala Ala Lys Ser Phe Glu Asp Leu Thr AspHis Pro Val 785 790 795 800 Thr Arg Ser Glu Lys Ala Ala Ser Phe Lys LeuGln Arg Gln Asn Arg 805 810 815 Val Asp Ser Lys Glu Thr Glu Cys 820 32097 DNA Homo sapiens CDS (1)..(2094) 3 atg agg cag tct ctc cta ttc ctgacc agc gtg gtt cct ttc gtg ctg 48 Met Arg Gln Ser Leu Leu Phe Leu ThrSer Val Val Pro Phe Val Leu 1 5 10 15 gcg ccg cga cct ccg gat gac ccgggc ttc ggc ccc cac cag aga ctc 96 Ala Pro Arg Pro Pro Asp Asp Pro GlyPhe Gly Pro His Gln Arg Leu 20 25 30 gag aag ctt gat tct ttg ctc tca gactac gat att ctc tct tta tct 144 Glu Lys Leu Asp Ser Leu Leu Ser Asp TyrAsp Ile Leu Ser Leu Ser 35 40 45 aat atc cag cag cat tcg gta aga aaa agagat cta cag act tca aca 192 Asn Ile Gln Gln His Ser Val Arg Lys Arg AspLeu Gln Thr Ser Thr 50 55 60 cat gta gaa aca cta cta act ttt tca gct ttgaaa agg cat ttt aaa 240 His Val Glu Thr Leu Leu Thr Phe Ser Ala Leu LysArg His Phe Lys 65 70 75 80 tta tac ctg aca tca agt act gaa cgt ttt tcacaa aat ttc aag gtc 288 Leu Tyr Leu Thr Ser Ser Thr Glu Arg Phe Ser GlnAsn Phe Lys Val 85 90 95 gtg gtg gtg gat ggt aaa aac gaa agc gag tac actgta aaa tgg cag 336 Val Val Val Asp Gly Lys Asn Glu Ser Glu Tyr Thr ValLys Trp Gln 100 105 110 gac ttc ttc act gga cac gtg gtt ggt gag cct gactct agg gtt cta 384 Asp Phe Phe Thr Gly His Val Val Gly Glu Pro Asp SerArg Val Leu 115 120 125 gcc cac ata aga gat gat gat gtt ata atc aga atcaac aca gat ggg 432 Ala His Ile Arg Asp Asp Asp Val Ile Ile Arg Ile AsnThr Asp Gly 130 135 140 gcc gaa tat aac ata gag cca ctt tgg aga ttt gttaat gat acc aaa 480 Ala Glu Tyr Asn Ile Glu Pro Leu Trp Arg Phe Val AsnAsp Thr Lys 145 150 155 160 gac aaa aga atg tta gtt tat aaa tct gaa gatatc aag aat gtt tca 528 Asp Lys Arg Met Leu Val Tyr Lys Ser Glu Asp IleLys Asn Val Ser 165 170 175 cgt ttg cag tct cca aaa gtg tgt ggt tat ttaaaa gtg gat aat gaa 576 Arg Leu Gln Ser Pro Lys Val Cys Gly Tyr Leu LysVal Asp Asn Glu 180 185 190 gag ttg ctc cca aaa ggg tta gta gac aga gaacca cct gaa gag ctt 624 Glu Leu Leu Pro Lys Gly Leu Val Asp Arg Glu ProPro Glu Glu Leu 195 200 205 gtt cat cga gtg aaa aga aga gct gac cca gatccc atg aag aac acg 672 Val His Arg Val Lys Arg Arg Ala Asp Pro Asp ProMet Lys Asn Thr 210 215 220 tgt aaa tta ttg gtg gta gca gat cat cgc ttctac aga tac atg ggc 720 Cys Lys Leu Leu Val Val Ala Asp His Arg Phe TyrArg Tyr Met Gly 225 230 235 240 aga ggg gaa gag agt aca act aca aat tactta ata gag cta att gac 768 Arg Gly Glu Glu Ser Thr Thr Thr Asn Tyr LeuIle Glu Leu Ile Asp 245 250 255 aga gtt gat gac atc tat cgg aac act tcatgg gat aat gca ggt ttt 816 Arg Val Asp Asp Ile Tyr Arg Asn Thr Ser TrpAsp Asn Ala Gly Phe 260 265 270 aaa ggc tat gga ata cag ata gag cag attcgc att ctc aag tct cca 864 Lys Gly Tyr Gly Ile Gln Ile Glu Gln Ile ArgIle Leu Lys Ser Pro 275 280 285 caa gag gta aaa cct ggt gaa aag cac tacaac atg gca aaa agt tac 912 Gln Glu Val Lys Pro Gly Glu Lys His Tyr AsnMet Ala Lys Ser Tyr 290 295 300 cca aat gaa gaa aag gat gct tgg gat gtgaag atg ttg cta gag caa 960 Pro Asn Glu Glu Lys Asp Ala Trp Asp Val LysMet Leu Leu Glu Gln 305 310 315 320 ttt agc ttt gat ata gct gag gaa gcatct aaa gtt tgc ttg gca cac 1008 Phe Ser Phe Asp Ile Ala Glu Glu Ala SerLys Val Cys Leu Ala His 325 330 335 ctt ttc aca tac caa gat ttt gat atggga act ctt gga tta gct tat 1056 Leu Phe Thr Tyr Gln Asp Phe Asp Met GlyThr Leu Gly Leu Ala Tyr 340 345 350 gtt ggc tct ccc aga gca aac agc catgga ggt gtt tgt cca aag gct 1104 Val Gly Ser Pro Arg Ala Asn Ser His GlyGly Val Cys Pro Lys Ala 355 360 365 tat tat agc cca gtt ggg aag aaa aatatc tat ttg aat agt ggt ttg 1152 Tyr Tyr Ser Pro Val Gly Lys Lys Asn IleTyr Leu Asn Ser Gly Leu 370 375 380 acg agc aca aag aat tat ggt aaa accatc ctt aca aag gaa gct gac 1200 Thr Ser Thr Lys Asn Tyr Gly Lys Thr IleLeu Thr Lys Glu Ala Asp 385 390 395 400 ctg gtt aca act cat gaa ttg ggacat aat ttt gga gca gaa cat gat 1248 Leu Val Thr Thr His Glu Leu Gly HisAsn Phe Gly Ala Glu His Asp 405 410 415 ccg gat ggt cta gca gaa tgt gccccg aat gag gac cag gga ggg aaa 1296 Pro Asp Gly Leu Ala Glu Cys Ala ProAsn Glu Asp Gln Gly Gly Lys 420 425 430 tat gtc atg tat ccc ata gct gtgagt ggc gat cac gag aac aat aag 1344 Tyr Val Met Tyr Pro Ile Ala Val SerGly Asp His Glu Asn Asn Lys 435 440 445 atg ttt tca aac tgc agt aaa caatca atc tat aag acc att gaa agt 1392 Met Phe Ser Asn Cys Ser Lys Gln SerIle Tyr Lys Thr Ile Glu Ser 450 455 460 aag gcc cag gag tgt ttt caa gaacgc agc aat aaa gtt tgt ggg aac 1440 Lys Ala Gln Glu Cys Phe Gln Glu ArgSer Asn Lys Val Cys Gly Asn 465 470 475 480 tcg agg gtg gat gaa gga gaagag tgt gat cct ggc atc atg tat ctg 1488 Ser Arg Val Asp Glu Gly Glu GluCys Asp Pro Gly Ile Met Tyr Leu 485 490 495 aac aac gac acc tgc tgc aacagc gac tgc acg ttg aag gaa ggt gtc 1536 Asn Asn Asp Thr Cys Cys Asn SerAsp Cys Thr Leu Lys Glu Gly Val 500 505 510 cag tgc agt gac agg aac agtcct tgc tgt aaa aac tgt cag ttt gag 1584 Gln Cys Ser Asp Arg Asn Ser ProCys Cys Lys Asn Cys Gln Phe Glu 515 520 525 act gcc cag aag aag tgc caggag gcg att aat gct act tgc aaa ggc 1632 Thr Ala Gln Lys Lys Cys Gln GluAla Ile Asn Ala Thr Cys Lys Gly 530 535 540 gtg tcc tac tgc aca ggt aatagc agt gag tgc ccg cct cca gga aat 1680 Val Ser Tyr Cys Thr Gly Asn SerSer Glu Cys Pro Pro Pro Gly Asn 545 550 555 560 gct gaa gat gac act gtttgc ttg gat ctt ggc aag tgt aag gat ggg 1728 Ala Glu Asp Asp Thr Val CysLeu Asp Leu Gly Lys Cys Lys Asp Gly 565 570 575 aaa tgc atc cct ttc tgcgag agg gaa cag cag ctg gag tcc tgt gca 1776 Lys Cys Ile Pro Phe Cys GluArg Glu Gln Gln Leu Glu Ser Cys Ala 580 585 590 tgt aat gaa act gac aactcc tgc aag gtg tgc tgc agg gac ctt tcc 1824 Cys Asn Glu Thr Asp Asn SerCys Lys Val Cys Cys Arg Asp Leu Ser 595 600 605 ggc cgc tgt gtg ccc tatgtc gat gct gaa caa aag aac tta ttt ttg 1872 Gly Arg Cys Val Pro Tyr ValAsp Ala Glu Gln Lys Asn Leu Phe Leu 610 615 620 agg aaa gga aag ccc tgtaca gta gga ttt tgt gac atg aat ggc aaa 1920 Arg Lys Gly Lys Pro Cys ThrVal Gly Phe Cys Asp Met Asn Gly Lys 625 630 635 640 tgt gag aaa cga gtacag gat gta att gaa cga ttt tgg gat ttc att 1968 Cys Glu Lys Arg Val GlnAsp Val Ile Glu Arg Phe Trp Asp Phe Ile 645 650 655 gac cag ctg agc atcaat act ttt gga aag ttt tta gca gac aac atc 2016 Asp Gln Leu Ser Ile AsnThr Phe Gly Lys Phe Leu Ala Asp Asn Ile 660 665 670 gtt ggg tct gtc ctggtt ttc tcc ttg ata ttt tgg att cct ttc agc 2064 Val Gly Ser Val Leu ValPhe Ser Leu Ile Phe Trp Ile Pro Phe Ser 675 680 685 att ctt gtc cat tgtgta acg tcg aaa tgc tga 2097 Ile Leu Val His Cys Val Thr Ser Lys Cys 690695 4 698 PRT Homo sapiens 4 Met Arg Gln Ser Leu Leu Phe Leu Thr Ser ValVal Pro Phe Val Leu 1 5 10 15 Ala Pro Arg Pro Pro Asp Asp Pro Gly PheGly Pro His Gln Arg Leu 20 25 30 Glu Lys Leu Asp Ser Leu Leu Ser Asp TyrAsp Ile Leu Ser Leu Ser 35 40 45 Asn Ile Gln Gln His Ser Val Arg Lys ArgAsp Leu Gln Thr Ser Thr 50 55 60 His Val Glu Thr Leu Leu Thr Phe Ser AlaLeu Lys Arg His Phe Lys 65 70 75 80 Leu Tyr Leu Thr Ser Ser Thr Glu ArgPhe Ser Gln Asn Phe Lys Val 85 90 95 Val Val Val Asp Gly Lys Asn Glu SerGlu Tyr Thr Val Lys Trp Gln 100 105 110 Asp Phe Phe Thr Gly His Val ValGly Glu Pro Asp Ser Arg Val Leu 115 120 125 Ala His Ile Arg Asp Asp AspVal Ile Ile Arg Ile Asn Thr Asp Gly 130 135 140 Ala Glu Tyr Asn Ile GluPro Leu Trp Arg Phe Val Asn Asp Thr Lys 145 150 155 160 Asp Lys Arg MetLeu Val Tyr Lys Ser Glu Asp Ile Lys Asn Val Ser 165 170 175 Arg Leu GlnSer Pro Lys Val Cys Gly Tyr Leu Lys Val Asp Asn Glu 180 185 190 Glu LeuLeu Pro Lys Gly Leu Val Asp Arg Glu Pro Pro Glu Glu Leu 195 200 205 ValHis Arg Val Lys Arg Arg Ala Asp Pro Asp Pro Met Lys Asn Thr 210 215 220Cys Lys Leu Leu Val Val Ala Asp His Arg Phe Tyr Arg Tyr Met Gly 225 230235 240 Arg Gly Glu Glu Ser Thr Thr Thr Asn Tyr Leu Ile Glu Leu Ile Asp245 250 255 Arg Val Asp Asp Ile Tyr Arg Asn Thr Ser Trp Asp Asn Ala GlyPhe 260 265 270 Lys Gly Tyr Gly Ile Gln Ile Glu Gln Ile Arg Ile Leu LysSer Pro 275 280 285 Gln Glu Val Lys Pro Gly Glu Lys His Tyr Asn Met AlaLys Ser Tyr 290 295 300 Pro Asn Glu Glu Lys Asp Ala Trp Asp Val Lys MetLeu Leu Glu Gln 305 310 315 320 Phe Ser Phe Asp Ile Ala Glu Glu Ala SerLys Val Cys Leu Ala His 325 330 335 Leu Phe Thr Tyr Gln Asp Phe Asp MetGly Thr Leu Gly Leu Ala Tyr 340 345 350 Val Gly Ser Pro Arg Ala Asn SerHis Gly Gly Val Cys Pro Lys Ala 355 360 365 Tyr Tyr Ser Pro Val Gly LysLys Asn Ile Tyr Leu Asn Ser Gly Leu 370 375 380 Thr Ser Thr Lys Asn TyrGly Lys Thr Ile Leu Thr Lys Glu Ala Asp 385 390 395 400 Leu Val Thr ThrHis Glu Leu Gly His Asn Phe Gly Ala Glu His Asp 405 410 415 Pro Asp GlyLeu Ala Glu Cys Ala Pro Asn Glu Asp Gln Gly Gly Lys 420 425 430 Tyr ValMet Tyr Pro Ile Ala Val Ser Gly Asp His Glu Asn Asn Lys 435 440 445 MetPhe Ser Asn Cys Ser Lys Gln Ser Ile Tyr Lys Thr Ile Glu Ser 450 455 460Lys Ala Gln Glu Cys Phe Gln Glu Arg Ser Asn Lys Val Cys Gly Asn 465 470475 480 Ser Arg Val Asp Glu Gly Glu Glu Cys Asp Pro Gly Ile Met Tyr Leu485 490 495 Asn Asn Asp Thr Cys Cys Asn Ser Asp Cys Thr Leu Lys Glu GlyVal 500 505 510 Gln Cys Ser Asp Arg Asn Ser Pro Cys Cys Lys Asn Cys GlnPhe Glu 515 520 525 Thr Ala Gln Lys Lys Cys Gln Glu Ala Ile Asn Ala ThrCys Lys Gly 530 535 540 Val Ser Tyr Cys Thr Gly Asn Ser Ser Glu Cys ProPro Pro Gly Asn 545 550 555 560 Ala Glu Asp Asp Thr Val Cys Leu Asp LeuGly Lys Cys Lys Asp Gly 565 570 575 Lys Cys Ile Pro Phe Cys Glu Arg GluGln Gln Leu Glu Ser Cys Ala 580 585 590 Cys Asn Glu Thr Asp Asn Ser CysLys Val Cys Cys Arg Asp Leu Ser 595 600 605 Gly Arg Cys Val Pro Tyr ValAsp Ala Glu Gln Lys Asn Leu Phe Leu 610 615 620 Arg Lys Gly Lys Pro CysThr Val Gly Phe Cys Asp Met Asn Gly Lys 625 630 635 640 Cys Glu Lys ArgVal Gln Asp Val Ile Glu Arg Phe Trp Asp Phe Ile 645 650 655 Asp Gln LeuSer Ile Asn Thr Phe Gly Lys Phe Leu Ala Asp Asn Ile 660 665 670 Val GlySer Val Leu Val Phe Ser Leu Ile Phe Trp Ile Pro Phe Ser 675 680 685 IleLeu Val His Cys Val Thr Ser Lys Cys 690 695 5 8 PRT Homo sapiens 5 LeuAla Gln Ala Val Arg Ser Ser 1 5 6 17 DNA Artificial Sequence Descriptionof Artificial Sequence mixed oligonucleotide primer 6 aartaygtna tgtaycc17 7 17 DNA Artificial Sequence Description of Artificial Sequence mixedoligonucleotide primer 7 ccrcartcrc aytcytc 17 8 6 PRT Homo sapiens 8Glu Glu Cys Asp Cys Gly 1 5 9 11 PRT Homo sapiens 9 Ile Ala Val Ser GlyAsp His Glu Asn Asn Lys 1 5 10

What is claimed is:
 1. An isolated and purified TACE polypeptide.
 2. Anisolated and purified polypeptide according to claim 1, that has amolecular weight of about 80 kD.
 3. An isolated and purified polypeptideaccording to claim 1, in non-glycosylated form.
 4. An isolated andpurified polypeptide according to claim 1 selected from the groupconsisting of a polypeptide comprising amino acids 18-Xaa of SEQ BD NO:2wherein Xaa ia an amino acid selected from the group consisting of aminoacids 671 through
 824. 5. Isolated and purified antibodies that bind tothe polypeptide according to claim
 1. 6. Isolated and purifiedantibodies according to claim 5, wherein the antibodies are monoclonalantibodies.
 7. A method for detecting the TACE-inhibiting activity of amolecule, comprising mixing said molecule with a substrate, incubating apolypeptide according to claim 1 with the mixture, andchromatographically determining the extent of substrate cleavage.
 8. Amethod for detecting the TACE-inhibiting activity of a moleculeaccording to claim 7, wherein the substrate comprises the amino acidsequence Leu-Ala-Gln-Ala-Val-Arg-Ser-Ser.
 9. A method of using apolypeptide according to claim 1 in a structure-based design of aninhibitor of said polypeptide, comprising the steps of deterininig thethree-dimensional structure of such polypeptide, analyzing thethree-dimensional structure for the likely binding sites of substrates,synthesizing a molecule that incorporates a predictive reactive site,and determining the polypeptide-inhibiting activity of the molecule. 10.A method for detecting the TNF-cleaving ability of a molecule,comprising incubating said molecule with a substrate that comprises theamino acid sequence Leu-Ala-Gln-Ala-Val-Arg-Ser-Ser, and determining theextent of substrate cleavage.
 11. An isolated nucleic acid selected fromthe group consisting of: (a) the coding region of a native mammalianTACE gene; (b) cDNA comprising nucleodides 52-2472 of SEQ ID NO: 1; (c)nucleic acid that is at least 80%o identical to the nucleic acid of (a)or (b) and that encodes a polypeptide that converts TNF-α from the 26 kDform to the 17 kD form; and (d) nucleic acid which is degenerate as aresult of the genetic code to a nucleic acid defined in (a), (b) or (c)and which encodes biologically active TACE.
 12. An isolated nucleic acidaccording to claim 1l, wherein the TACE is human TACE.
 13. An isolatednucleic acid according to claim 11, which encodes a polypeptidecomprising amino acids 18-671 of SEQ ID NO:2.
 14. An expression vectorthat directs the expression of a nucleic acid sequence according toclaim
 11. 15. A host cell transfected or transformed with the expressionvector according to claim
 11. 16. A process for producing a TACEpolypeptide, comprising culturing a host cell according to claim 15under conditions promoting expression, and recovering the polypeptidefrom the culture medium
 17. A method of inhibiting the cleavage of TNF-αfrom cell membranes in a mammal comprising administering to such mammalan effective amount of a compound that inhibits the TNF-α proteolyticactivity of an enzyme comprising the sequence of amino acids 18-671 ofSEQ ID NO:2.
 18. A method of inhibiting TNF-α cleavage from cellmembranes comprising blocking the binding of TNF-α with an enzyme havingthe sequence of amino acids 18-671 of SEQ ID NO:2.
 19. A method fortreating a mammal having a disease characterized by an overproduction oran upregulated production of TNF-α , comprising administering to themammal a composition comprising an amount of a compound that effectivelyinhibits the TNF-α proteolytic activity of an enzyme comprising thesequence of amino acids 18-671 of SEQ ID NO:2.